Coferons and methods of making and using them

ABSTRACT

A monomer useful in prepaπng therapeutic compounds includes a diversity element which potentially binds to a target molecule with a dissociation constant of less than 300 11 M and a linker element connected to the diversity element The linker element has a molecular weight less than 500 daltons, is connected, directly or indirectly through a connector, to said diversity element, and is capable of forming a reversible covalent bond or noncovalent interaction with a binding partner of the linker element The monomers can be covalently or non-covalently linked together to form a therapeutic multimer or a precursor thereof.

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 61/043,537, filed Apr. 9, 2008, which is herebyincorporated by reference in its entirety.

This invention was made with government support under Public HealthService grant AI062579-03 from the National Institute of Allergy andInfectious Diseases and Grant No. CA65930-08 from the National CancerInstitute. The government has certain rights in this invention.

FIELD OF THE INVENTION

The present invention is directed to coferons and methods of making andusing them.

BACKGROUND OF THE INVENTION

Cancers arise due to mutations or dysregulation of genes involved in DNAreplication and repair, cell cycle control, anchorage independentgrowth, angiogenesis, apoptosis, tissue invasion, and metastasis(Hanahan, D. et al., Cell 100(1): 57-70 (2000)). These processes arecontrolled by networks of genes in the p53, cell cycle, apoptosis, Wntsignaling, RPTK signaling, and TGF-beta signaling pathways. Such genesand their protein products are the targets of many current anddeveloping therapies.

Signaling pathways are used by cells to generate biological responses toexternal or internal stimuli. A few thousand gene products control bothontogeny/development of higher organisms and sophisticated behavior bytheir many different cell types. These gene products work in differentcombinations to achieve their goals, and do so through protein-proteininteractions. The evolutionary architecture of such proteins is throughmodular protein domains that recognize and/or modify certain motifs. Forexample, different tyrosine kinases (such as Abl) will add phosphategroups to specific tyrosines inbedded in particular peptide sequences,while other enzymes (such as PTEN) act as phosphatases to remove certainsignals. Proteins and other macromolecules may also be modified throughmethylation, acetylation, sumolation, ubiquitination, and these signalsin turn are recognized by specific domains that activate the next stepin the pathway. Such pathways usually are initiated through signals toreceptors on the surface, which move to intracellular proteininteractions and often lead to signaling through transcription factorinteractions that regulate gene transcription. For example, in the Wntpathway, Wnt interacts with the Frizzled receptor, signaling throughDisheveled, which inhibits the Axin-APC-GSK3 complex, which binds tobeta-catenin to inhibit the combination of beta-catenin with TCF4,translocation of this complex into the nucleus, and activation of Myc,Cyclin D, and other oncogenic protein transcription (Polakis, P. et al.,Genes Dev 14(15):1837-1851 (2000); Nelson, W. J. et al., Science303(5663):1483-1487 (2004)). Signaling may also proceed from the nucleusto secreted factors such as chemokines and cytokines (Charo, I. F. etal., N Engl J Med 354(6):610-621 (2006)). Protein-protein andprotein-nucleic acid recognition often work through protein interactionsdomains, such as the SH2, SH3, and PDZ domains. Currently, there areover 75 such motifs reported in the literature (Hunter, et. al., Cell100:113-127 (2000); Pawson et. al., Genes & Development 14:1027-1047(2000)). These protein-interaction domains comprise a rich opportunityfor developing targeted therapies.

Cancer therapies may be divided into two classical groups: (i) smallmolecule drugs such as Gleevec that bind into a compact pocket, and (ii)antibody therapeutics such as herceptin which binds and inhibits theHER-2/neu member of the epidermal growth factor receptor (EGFR) family.Antibody and protein therapeutics work by binding over an extended areaof the target protein. Antibodies fight cancers by inducing apoptosis,interfering with ligand-receptor interactions, or preventing expressionof proteins required for tumor growth (Mehren et al., Ann Rev. Med.54:343-69 (2003)). Additional successful cancer antibody therapeuticsinclude Rituximab, an anti CD20 antibody, Erbitux (cetuximab) targetedto EGFR, and Avastin (bevacizumab) which interferes with vascularendothelial growth factor (VEGF) binding to its receptor (Mehren et al.,Ann Rev. Med. 54:343-69 (2003)). Except for the skin rash associatedwith EGFR receptor antibodies (which ironically correlates withefficacy), antibody therapies are generally well tolerated and do nothave the side-effects associated with traditional chemotherapy.

Antibodies achieve their extraordinary specificity through the diversitygenerated in their complementarity-determining regions (“CDR's”). An IgGantibody binding surface consists of three CDRs from the variable heavychain paired with three CDRs from the variable light chain domain. EachCDR consists of a loop of around a dozen amino acid residues, whosestructure binds to the target surface with nanomolar affinity (Laune,et. al., J. Biol. Chem. 272:30937-30944 (1997); Monnet, et al., J. Biol.Chem. 274:3789-3796 (1999)). Thus, antibodies achieve their specificityby combining multiple weak interactions across a generally flat surfaceof approximately 1200-3000 Å². Monoclonal antibodies may be readilygenerated to most proteins, and artificial antibodies screened for usingin vitro phage or bacterial systems (Mehren et al., Ann Rev. Med.54:343-69 (2003)). Mouse monoclonal antibodies may be “humanized” toreduce development of undesired human antimouse antibodies. Limitationsof using monoclonal antibodies include production of anti-idiotypicantibodies, disordered tumor vasculature, increased hydrostatic pressurewithin tumor, and heterogeneity of surface antigen within tumors. Due tothese barriers, it takes 2 days for an IgG antibody to travel 1 mm and7-8 months to travel 1 cm into a tumor (Mehren et al., Ann Rev. Med.54:343-69 (2003)). Smaller variations of the IgG motif's have beenengineered, including scFv and Affibodies (Eliasson, M. et al., JImmunol 142(2):575-581 (1989); Gunneriusson, E. et al., J Bacteriol178(5):1341-1346 (1996); Nord, K. et al., Nat Biotechnol 15(8):772-777(1997)), and these have improved tumor penetration by cutting downpenetration time in about half.

Antibodies can achieve tighter binding and higher specificity than anyartificially synthesized therapy. Nevertheless, antibody therapies arelimited to interfering with protein-protein interactions or proteinreceptor activity that are on the surface of tumors or circulatingtargets, cannot be ingested orally, and are not able to use theirextraordinary specificity to inhibit intracellular protein signaling.

On the other end of the spectrum are small molecule drugs. These havethe advantages of being orally active, being sufficiently small enough(usually with a molecular weight <750) to diffuse across cell membranes,and binding tightly into compact binding pockets used by all enzymes tobind their substrates (or interfering with macromolecular machinery usedin cellular processes) (Landry, Y., et al., Fundam Clin Pharmacol22(1):1-18 (2008); Duarte, C. D., et al., Mini Rev Med Chem7(11):1108-1119 (2007); Amyes, T. L., et al., ACS Chem Biol2(11):711-714 (2007)). Recently, the field of combinatorial chemistryhas greatly improved the ability of chemists to identify lead moleculesthat bind and inhibit specific protein targets (Dolle, et al., J.Combinatorial Chem. 6(5):597-635 (2005)).

Thus, current drug design and drug therapy approaches do not address theurgent need to find drugs which interfere with intracellularprotein-protein interactions, or protein signaling. Antibodies have therequired specificity to distinguish among closely related proteinsurfaces, yet are too large to be taken orally or enter cells. Orallyactive pharmaceuticals are too small (i.e. have a molecular weight lessthan 750) to disrupt protein-protein surface interactions (generallyflat, and over 1200-3000 Å²).

Attempts to identify small molecule drugs that bind over an extendedarea have mostly been limited to traditional targets containing at leastone compact binding site. One approach is based on: (i) preparing a setof potential binding elements where each molecule has a common chemicallinkage group; (ii) identifying all binding elements that inhibit evenweakly the target enzyme; (iii) preparing a combinatorial library of allthe winning binding elements connected by a common chemical linkagegroup and a series of flexible linkers; and (iv) screening thecombinatorial library to identify the tightest binding compound drugs.This approach was used to identify a small molecule inhibitor of thec-Src tyrosine kinase (Maly, et. al., Proc. Nat'l Acad. Sci. USA 97:2419-2424 (2000)) as well as the tyrosylprotein sulfotransferase (Kehoe,et al., BioOrg & Medicinal Chem. Lett. 12:329-332 (2002)). One flaw inthis approach is that the initial screen finds mostly molecules thatbind within the initial pocket, but the final product needs to have bothbinding elements bind with high affinity. Thus, the success of the aboveapproach was the result of a fortuitous alternative binding of one ofthe elements identified in the initial screen. A second disadvantage isthe need to screen each of the potential combinatorial library elementsindividually.

To overcome the limitation of testing various combinations of ligandsand connectors individually, Lehn and coworkers developed dynamiccombinatorial chemistry (“DCC”) as a new means for drug discovery (Lehn,et. al., Science 291:2331-2332 (2001); Ramstrom, et. al., Nat. Rev. DrugDiscovery 1:26-36 (2002)). In this approach, potential ligand moleculesform reversible adducts to different bifunctional connector molecules,and these interconnections are in continuous exchange with each other.When the enzyme target is added, the best bound library constituent isselected from all the possible combinations, allowing for identificationof the active species. Using 16 hydrazides, 2 monoaldehydes, and 3dialdehydes, 440 different combinations were formed and selected againstthe bifunctional B. subtilis HPr. kinase/phosphatase (Bunyapaiboonsri,et. al., J. Med. Chem. 46:5803-5811 (2003)). Improvement in synthesisand spatial identification of specific library members is achieved byusing resin-bound DCC approaches (McNaughton, et. al., Organic Letters8:1803-1806 (2006)).

The use of DNA to encode self-assembling chemical (ESAC) libraries hasextended the potential for dynamic combinatorial chemistry drugdiscovery (Melkko et al., Nature Biotech, 22:568-574 (2004)). The DNAstrands are partially complementary to allow for reversible binding toeach other under standard incubation conditions and also contain barcodes to identify the ligand element. After using DCC to select for thetightest binding combinations, and identification of ligands based ontheir DNA code, the ligands are resynthesized with a variety of spacersto identify the tightest binding tethered combinations. This approachwas used to find binding molecules with nanomolar affinities to serumalbumin, carbonic anhydrase, streptavidin, and trypsin respectively(Melkko et al., Nature Biotech, 22:568-574 (2004); Dumelin et al.,Bioconjugate Chem. 17:366-370 (2006); Melkko et al., Angew. Chem.46:4671-4674 (2007)). One disadvantage of this approach is the widefootprint of about 15.4 Angstroms introduced by using double-strandedDNA as the dynamic combinatorial chemistry element, separating theligands by a considerable distance, and requiring a higher MW tether toreestablish tight binding affinities.

In an inversion of the standard small-molecule drug binding within acompact binding pocket in the target enzyme, the macrocycle vancomycinbinds to its L-Lys-D-Ala-D-Ala tripeptide target by forming a dimer thatsurrounds the tripeptide. By using the actual target to acceleratecombinatorial synthesis of vancomycin and vancomycin analogue dimers,tethered dimers were isolated with tighter affinities and in vitroactivity against some vancomycin resistant bacterial strains (Nicolaouet al., Angew. Chem. 39:3823-3828 (2000)). It is unlikely that thesederivatives would be orally active due to their high molecular weightand potential for disulfide dimers to be reduced to monomers within thebloodstream.

Many receptors (for example, the erythropoietin receptor) are activatedby ligand-induced homodimerization, which leads to internal cellularsignals. By using bi- or multi-functional connectors to link ligandmolecules to form dimers, trimers, and tetramer libraries, a number ofsmall molecule agonists could be isolated that assisted inerythropoietin receptor homodimerization (Goldberg et. al., J. Am. Chem.Sec. 124:544-555 (2002)). These molecules demonstrate the ability ofmulti-ligand drugs to influence protein-protein interactions, in amanner that mimics the natural activity of cytokines and chemokines.

Sharpless and coworkers have identified reactions that occur readilywhen the constituent chemical linkage groups are brought in closeproximity with each other, termed “click chemistry” (Kolb, et. al., DrugDiscovery Today 8:1128-1137 (2003)). By adding various ligands connectedto these reactive groups (such as an azide on one set of ligands andacetylene on the other ligands) and combining these library compounds insolution in the presence of enzyme targets, highly potent inhibitorsform, for example for the acetylcholine esterase or the HIV protease(Kolb et. al., Drug Discovery Today, 8:1128-1137 (2003); Brik et. al.,Chem. BioChem 4:1246-1248 (2003); Whiting, et. al., Angew. Chem. Int.Ed. 45:1435-1439 (2006); Lewis et. al., Angew Chem 41:1053-1057 (1002);Bourne et. al., Proc. Nat'l Acad. Sci. USA 101:1449-1454 (2004)). Inshort, the target enzyme acts as a catalyst for the proximal ligation ofits own inhibitor. The advantage of this approach is the enrichment ofthe best binding compound in a single step.

An elegant approach to finding low molecular weight ligands that bindweakly to targeted sites on proteins was developed by Wells andcoworkers (Erlanson et. al., Proc. Nat'l Acad. Sci. USA 97:9367-9372(2000); Thanos, et. al., J. Am. Chem. Sco. 125:15280-15281 (2003);Erlanson et. al., Nature Biotechnology 21:308-314 (2003); Buck et. al.,Proc. Nat'l Acad. Sci. USA 102:2719-2724 (2005)). A native or engineeredcysteine in a protein is allowed to react reversibly with a smalllibrary of disulfide-containing molecules. The process of dynamiccombinatorial chemistry takes place as the most stable molecules areenriched on the surface of the protein target. These are then readilyidentified by mass spectroscopy, and serve as lead compounds for furthermodification.

Dynamic combinatorial or “click” chemistry increases yields ofappropriate binding ligand combinations, but still requires enzymaticassays. The disadvantages of these approaches are that they are limitedto enzymes with one or more deep binding pockets, where knowledge of atleast one potential ligand is often needed. Further, the starting blocksare not readily available and require independent synthesis for eachdiversity element or ligand to be tested. The chemical linkage groupsused for click chemistry are not suitable for use in vivo as they wouldreact readily and irreversibly with cellular components. The reactionsneed to take place with sufficient efficiency and at a large enoughscale such that the enzyme selected inhibitor is synthesized insufficient amounts to allow for purification and identification of thecorrect product. This last constraint limits the number of ligands thatmay be screened in a single assay, and limits the throughput of theseapproaches.

Several groups have recognized that macrocycles provide an opportunityfor recognition of extended binding motifs within targets. Several ofthese are orally active, despite having molecular weight beyond thetraditional 750 cutoff. These include cyclosporin (molecular weight1202.64), rapamycin (molecular weight 914.2), tacrolimus (molecularweight 822.03), erythromycin (molecular weight 733.94), azithromycin(molecular weight 748.88), and clarithromycin (molecular weight 747.9).Note that although vancomycin (molecular weight 1485.74) is used orallyfor treatment of gastrointestinal infections, it is not absorbed intothe body. Cyclosporin is the largest of the groups listed above andillustrates a few features common to these drugs. Their cyclic naturereduces entropic loss upon binding and the extended structure allows forenhanced binding. Cyclosporin has torroidal flexibility, allowing it tobring its polar side-chains into the interior so the outside is nonpolarand this allows for transfer across membranes. Likewise, the drug is instructural equilibrium with its polar conformer, allowing for binding toits target.

As promising as macrocycle and synthetic peptide mimetics are for leaddrug candidates, it is not trivial to use synthetic chemistry togenerate sufficient diversity required for high affinity binding toextended binding sites in target proteins. Two groups have sought toaddress this issue using DNA encoded approaches with evolutionaryselection. In the first approach, a functional group is attached to along DNA barcode sequence containing multiple zip-codes (Halpin, D. R.et al., PLoS Biol 2(7):E173 (2004); Halpin, D. R. et al., PLoS Biol2(7):E174 (2004); Halpin, D. R. et al., PLoS Biol 2(7):E175 (2004)). Themolecules are equilibrated with a set of columns (e.g., 10 columns),containing beads with complementary zip-code sequences. DNAhybridization captures library members containing the complementaryzip-code sequence on their DNA tag. The library members are eluted intoseparate new chambers and reacted with a bifunctional moiety (forexample, a protected amino acid residue) that corresponds to the givenzip-code. The library members are then re-pooled, and then rerouted tothe next series of columns. This process was repeated through severalrounds to generate 10⁶ pentapeptides. After only two rounds oftranslation, selection with an antibody to the pentapeptide enkephalin,and amplification, the library converged on enkephalin and slightvariants. Potential disadvantages of this approach are the need for DNAencryption strands of 200 or more bases. In the second approach, abifunctional group is attached to a DNA template sequence containingadjacent zipcode sequences (Calderone, C. T. et al., Angew Chem Int EdEngl 44(45):7383-7386 (2005); Sakurai, K. et al., J Am Chem Soc127(6):1660-1661 (2005)). The DNA sequence serves as a template foradding bifunctional moieties to one end of the bifunctional group on theDNA tag. Each bifunctional moiety (for example, a protected amino acidresidue) is attached to a complementary zip-code DNA molecule, whichhybridizes on the DNA template containing the original bifunctionalgroup. This hybridization increases the local concentration of thereactant to such an extent that it can drive synthesis to very highyields. This method does not require split-pooling techniques. If 4 setsof 10 each bifunctional moieties are added, this will result in 10,000diversity elements in the library. At the end of the synthesis, the lastamino acid residue may be reacted with the other end of the originalbifunctional group to create a circular diversity element. In thisversion, the identity of the diversity element is defined by the zipcodesequences in the DNA template. It may be identified by PCR amplificationand sequencing. Further, the PCR amplicons may serve as startingtemplates for a new round of translation, selection, and amplification,allowing for application of evolutionary principles to synthesize highaffinity binding elements. However, the extent of diversity elementssynthesized by the above two approaches are still several orders ofmagnitude lower than the diversity and affinity achieved by just asingle CDR loop from an antibody molecule.

Several groups have investigated the ability of small molecules tointeract with each other or encircle other small molecule targets; theseare known as “guest-host” interactions or artificial receptors. However,these compounds are not suitable, because they are not of low enoughmolecular weight or interact under non-physiological conditions or wouldbe too reactive with other intracellular molecules.

A common approach to designing artificial receptors is to construct a“molecular tweezer”, consisting of a two armed structure joined by aconformationally restricted linker, such that the two arms point in thesame direction (analogous to a tweezer). These “host” structures areoften designed with a dye or on a bead, and then screened for binding ofthe “guest”, most often a tri-peptide, again with either a dye or on abead. (Shao et. al., J. Org. Chem. 61:6086-6087 (1996); Still et. al.,Acc. Chem. Res. 29:155-163 (1996); Cheng, et. al., J. Am. Chem. Soc.118:1813-1814 (1996); Jensen et. al., Chem. Eur. J. 8:1300-1309 (2002)).In a variation of this theme, binding of the peptide displaces aquenched fluorescent group from the host pocket, thus creating afluorescent signal upon binding (Chen, et. al., Science 279:851-853(1998); Iorio et. al., Bioorganic & Medicinal Chem. Lett. 11:1635:1638(2001)). Rigid diketopiperazine backbone receptors with tri-peptide armshave demonstrated both tight binding, as well as how small structuralchanges in the backbone significantly reduce that binding (Wennemers etal., Chem. Eur. J. 7:3342-3347 (2001); Conza et. al., J. Org. Chem.67:2696-2698 (2002); Wennemers et al., Chem. Eur. J. 9:442-448 (2003)).Unsymmetrical tweezer and one-armed receptor hosts have been designed tomimic vancomycin binding of an L-Lys-D-Ala-D-Ala tripeptide guest(Shepard et al., Chem. Eur. 1 12:713-720 (2006); Schmuck et al., Chem.Eur. J. 12:1339-1348 (2006)). Other host-guest systems includenapthalene-spaced tweezers and cyanobenzene derivatives (Schaller etal., J. Am. Chem. Soc. 129:1293-1303 (2007)). In some of the examplesabove, the selection was performed in organic solvents, and, in allcases, at least one of the entities had a molecular weight in excess of400 and often in excess of 800. Thus, these examples would not besuitable for lead molecules.

Another approach to designing low molecular weight affinity binders isto use phage display. This approach was used to find peptides from 9-13mers that bind fluorescent dyes; however, only one of these retainedsufficient affinity to bind a dye when resynthesized outside the contextof the phage protein (Rozinov et. al., Chemistry & Biology 5:713-728(1998), Marks, et. al., Chemistry & Biology 11:347-356 (2004)). Othergroups have used phage display to design synthetic peptides 8-12 mersthat bind biotin (Saggio et. al., Biochem. J. 293:613-616 (1993)),camptothecin (Takakusagi et al., Bioorganic & Medicinal Chem. Lett.15:4850-4853 (2005)), as well as doxorubicin and other hydrophobiccancer drugs (Popkov et al, Eur. J. Biochem. 251:155-163 (1998)). In allthese cases, the fluorescent dye or similarly hydrophobic guest moietyis held in place by a pocket comprised from hydrophobic amino acids, andthen additional residues may provide further stability. Since thepeptides have molecular weights ranging from about 900 to about 1500,they are too large and not suitable for lead molecules.

Thus, there is a need to design new small molecules that associate withgood affinities for one another under physiological conditions. Thepresent invention is directed to overcoming this deficiency in the art.

SUMMARY OF THE INVENTION

One aspect of the present invention is directed to a monomer useful inpreparing therapeutic compounds. The monomer includes a diversityelement which potentially binds to a target molecule with a dissociationconstant of less than 300 μM and a linker element connected, directly orindirectly through a connector, to said diversity element. The linkerelement has a molecular weight less than 500 daltons and is capable offorming a reversible covalent bond or non-covalent interaction with abinding partner of said linker element with a dissociation constant ofless than 300 μM with or without a co-factor under physiologicalconditions.

Another aspect of the present invention relates to a therapeuticmultimer precursor. The therapeutic multimer precursor includes aplurality of covalently or non-covalently linked monomers. Each monomercomprises a diversity element which potentially binds to a targetmolecule with a dissociation constant less than 300 μM, a linkerelement, and an encoding element. The linker element has a molecularweight less than 500 daltons and is capable of forming a reversiblecovalent bond or non-covalent tight interaction with a binding partnerof said linker element with a dissociation constant less than 300 μM,with or without a co-factor, under physiological conditions. Thediversity element and the linker element for each monomer are connectedtogether, directly or indirectly through a connector, and the pluralityof monomers are covalently bonded together or non-covalently linkedtogether through their linker elements. The diversity elements for theplurality of monomers bind to proximate locations of the targetmolecule.

Yet a further embodiment of the present invention is directed to amethod of screening for therapeutic compound precursors which bind to atarget molecule associated with a condition. This method includesproviding a plurality of monomers. Each monomer comprises a diversityelement which potentially binds to a target molecule with a dissociationconstant less than 300 μM and a linker element capable of forming areversible covalent bond or non-covalent interaction with a bindingpartner of the linker element with or without a co-factor underphysiological conditions. The linker has a molecular weight of less than500 daltons. The diversity element and said linker element of eachmonomer are joined together directly or indirectly through a connector.The plurality of monomers are contacted with the target molecule underconditions effective to permit diversity elements able to bind to thetarget molecule to undergo such binding. The monomers are then subjectedto reaction conditions effective for the linker elements of differentmonomers to undergo covalent bonding or non-covalent interactions toform therapeutic multimer precursors, either before, after, or duringthe contacting step. The monomers forming each therapeutic multimerprecursor are then identified.

Another embodiment of the present invention involves a method ofscreening for linker elements capable of binding to one another. Thismethod includes providing a first and a second set of monomers. Each ofthe monomers in the first set comprise a linker element, having amolecular weight of less than 500 daltons and being capable of forming areversible covalent bond or non-covalent interaction with a bindingpartner of said linker element, with or without a co-factor underphysiological conditions and a sulfhydryl group. The linker element,with or without a co-factor, and the sulfhydryl group for each monomerof the first set of monomers are coupled together. Each of the monomersin the second set comprise a linker element capable of forming areversible covalent bond or reversible non-covalent bonds with a bindingpartner of the linker element under physiological conditions, an encodedbead, and a sulfhydryl group. The linker element, the encoded bead, andthe sulfhydryl group for each monomer of the second set of monomers arecoupled together. The first and second sets of monomers are contactedwith one another under physiological conditions so that monomers fromthe first set of monomers and monomers from the second set of monomersbind together to form multimers linked together by disulfide bondsformed from their sulfhydryl groups and, potentially, covalent bonds ornon-covalent interactions between their linker elements. The dimerswhere the linker elements from the monomers of the first and second setsof monomers are covalently bound or non-covalently linked together arethen identified as being candidate multimers. The linker elements fromthe first and second monomers that are covalently bound ornon-covalently joined together are then identified in the candidatemultimers.

An additional embodiment of the present invention relates to atherapeutic multimer which includes a plurality of covalently ornon-covalently linked monomers. Each monomer comprises a diversityelement which binds to a target molecule with a dissociation constant ofless than 300 μM and a linker element having a molecular weight lessthan 500 daltons, and capable of forming a reversible covalent bond ornon-covalent tight interaction with a binding partner of the linkerelement with a dissociation constant less than 300 μM, with or without aco-factor, under physiological conditions. The diversity element and thelinker element are joined together for each monomer, directly orindirectly through a connector. The plurality of monomers are covalentlybonded together or non-covalently linked together through their linkerelements, and the diversity elements for the plurality of monomers bindto proximate locations of the target molecule.

Another embodiment of the present invention relates to a plurality oftherapeutic monomers capable of combining to form a therapeuticmultimer. Each monomer includes a diversity element which binds to atarget molecule and a linker element having a molecular weight less than500 daltons and capable of forming a covalent bond or non-covalent tightinteraction with a binding partner of the linker element with adissociation constant less than 300 μM, with or without a co-factor,under physiological conditions. The diversity element, which has adissociation constant less than 300 μM, and the linker element areconnected together directly or indirectly through a connector for eachmonomer. A plurality of monomers are capable of covalently bondingtogether or being non-covalently linked together through their linkerelements, and the diversity elements for the plurality of monomers bindto proximate locations of the target molecule.

The linker elements of the present invention are small molecules thatexclusively associate with each other in vivo and do not react withcellular components. Each linker element has attachment points forintroducing diverse ligands and individual DNA encryption elements. Theyare compatible with “click chemistry” and DNA-templated synthesis. Theassociation between the linker elements is reversible, allowing fordynamic combinatorial chemistry selection of the best ligands. Thelinker elements allow in vivo assembly of multiple small ligands toproduce structures having a molecular weight up to about 4800 andpotentially disrupt protein-protein interactions.

The linker elements of the present invention have the potential tomodulate or inhibit protein-protein signaling. The combined size of thelinker element-ligand dimers and multimers provides sufficient surfacearea to interact with protein surfaces with increased selectivity andreduced toxicity. Directed evolution selects for tightest binding leadcompounds, with the potential to drive affinities to sub-nmol range.

The present invention is directed to a novel class of drug molecules(referred to here as coferons) that assemble in vivo. A coferon monomeris composed of a diversity element or ligand that binds to the targetand a dynamic combinatorial chemistry element herein termed a linkerelement. The linker element of one coferon monomer may reversiblycombine with the linker element of another coferon monomer in vivo toform a coferon dimer. In some cases, the linker element binding to eachother may be essentially irreversible. In additional cases, the linkerelements bind to each other with the aid of a co-factor. In other cases,the linker elements are in a precursor form, and are activated uponentering the body or cells. The linker elements bind to each otherthrough hydrophobic, polar, ionic, hydrogen bonding, and/or reversiblecovalent interactions. In the presence of the target, the combinationsof multiple (weak) interactions between the diversity element of onecoferon monomer and a target protein, the diversity element of a secondcoferon monomer and the target protein, as well as the two coferons witheach other combine to produce a tight binding coferon dimer with highlyspecific binding to its target. The concept may be extended to includemultimer coferons and multimer targets.

Since coferon monomers associate in a reversible manner, the principalsof dynamic combinatorial chemistry selection may be used to identify thebest ligands for each target in vitro. Combining two coferon libraries,for example with 10⁴ diversity elements provides the opportunity toscreen 10⁸ combinations simultaneously. Use of repeated synthesis,selection, and amplification strategies will allow for evolutionaryselection of coferon dimers with nanomolar and even subnanomolar bindingaffinities. The combined size of linker element dimers providessufficient surface area to interact with extended binding proteinsurfaces. Nevertheless, since coferon assembly on the target isdependent on multiple additive interactions, false binding to incorrectproteins will be rare (and can be selected against), and, thus, suchdrugs should have minimal to no off-target toxicity. Use of circularpeptide and peptide analogue containing diversity elements will alsoallow for switching between polar and non-polar conformers for easiertransport across membranes. Coferon monomers may be designed to have amolecular weight of less than 1000, allowing them to be orally active,penetrate deeply into tumors, and cross membrane barriers to enterinside cells—significant advantages over antibodies—while providingequal specificity.

The key to the linker elements is identifying small molecules (withmolecular weights preferably within the range of 45 to 450 daltons) thatassociate with good affinities for one another in vivo, and preferablyassociate exclusively with each other in vivo. They should not reactwith cellular components. The more sophisticated linker elementsdescribed below help catalyze formation of reversible covalent bondswhen binding to each other under physiological conditions. The varietyof coferon designs may be expanded by uncoupling the screening processfor diversity element ligands from the final coferon structure used inthe drug. This allows the use of linker elements in the final drug whosebinding is essentially irreversible. Essentially irreversible linkerelements are generally, but not limited to, linker elements that mayassociate slowly or even very slowly, either in the absence or presenceof the target. However, once formed, such linker elements essentially donot dissociate.

Even though each individual bond between two linker elements may bereversible, once both bonds are established, reversal of one bond stillkeeps the two reactants in such close proximity that they will de factoreform the bond again. Certain linker elements may be reversible undersome conditions (used during screening), yet essentially irreversibleunder other conditions, for example when formulated in the final drug.For those linker elements that have the potential to combineirreversibly during formulation, or alternatively in the body prior toentering the target cells, the reactive groups may be protected andrendered unreactive. Upon entering the target cells, the protectinggroup may be removed by cellular processes, such as disulfide reductionto the thiol by intracellular glutathione, enzymatic cleavage (i.e.esterase), or pH change (if entry is via endosomes or linker elementsenter lysosomal compartments) or simply by reversible dissociation upondilution into the blood stream (i.e. reversible alcohol protection of areactive boronate group). Linker elements that are essentiallyirreversible under dynamic combinatorial chemistry (DCC) screeningconditions may be rendered reversible using a new approach describedherein, which we term “cyclic combinatorial chemistry” (C3) screening.

Subcategories of coferons will be divided into those that bind theirtarget as dimers and those that work as multimers. Some of the coferonsmay be easily modified to bring two dimers together in a head-to-headfashion to create tetramers or even higher-order coferon multimers.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic drawing of the components used in Coferon monomer.

FIGS. 2.1A to 2.1K show the variations of the components of coferon drugdesign. FIG. 2.1A is a schematic drawing of coferon monomers inaccordance with the present invention attached to encoded beads viaconnectors. FIG. 2.1B is a schematic drawing of a coferon monomer inaccordance with the present invention with a DNA barcode attachedthrough a connector. FIG. 2.1C is a schematic drawing of a coferon dimerattached to an encoded bead via a connector to one monomer, with a DNAbarcode attached to the other monomer. FIG. 2.1D is a schematic drawingof a coferon dimer, with DNA barcodes attached to each monomer via theconnectors. FIG. 2.1E is a schematic drawing of a coferon dimer withconnectors pursuant to the present invention. FIG. 2.1F is a schematicdrawing of coferon monomers in accordance with the present inventionattached to an encoded bead via the linker element. FIG. 2.1G is aschematic drawing of a coferon monomer in accordance with the presentinvention with a DNA barcode attached to the linker element. FIG. 2.1His a schematic drawing of a coferon dimer attached to an encoded beadvia the linker element to one monomer, with a DNA barcode attached tothe other monomer. FIG. 2.1I is a schematic drawing of a coferon dimer,with an encoded bead attached one monomer via its linker element. FIG.2.1J is a schematic drawing of a coferon dimer, with DNA barcodesattached to each monomer via the linker elements. FIG. 2.1K is aschematic drawing of a coferon dimer pursuant to the present invention.

FIGS. 2.2L to 2.2S show additional variations of the components ofcoferon drug design. FIG. 2.2L is a schematic drawing of a coferon dimerattached to an encoded bead via a connector to one monomer, with a DNAbarcode attached to the other monomer. The linker elements bind to eachother via a cofactor, such as a metal ion (blue dot). FIG. 2.2M is aschematic drawing of a coferon dimer, with DNA barcodes attached to eachmonomer via the connectors. The linker elements bind to each other via acofactor, such as a metal ion. FIG. 2.2N is a schematic drawing of acoferon dimer, which bind to each other via a cofactor, pursuant to thepresent invention. FIG. 2.2O is a schematic drawing of a coferon dimerattached to an encoded bead via a connector to one monomer. The linkerelements bind to each other via a cofactor, such as a metal ion. FIG.2.2P is a schematic drawing of a coferon dimer attached to an encodedbead via the linker element of one monomer. The linker elements bind toeach other via a cofactor, such as a metal ion. FIG. 2.2Q is a schematicdrawing of a coferon dimer attached to an encoded bead via the linkerelement to one monomer, with a DNA barcode attached to the othermonomer. The linker elements bind to each other via a cofactor, such asa metal ion. FIG. 2.2R is a schematic drawing of a coferon dimer, withDNA barcodes attached to each monomer via the linker elements, whichbind to each other via a cofactor, such as a metal ion. FIG. 2.2J is aschematic drawing of a coferon dimer, which bind to each other via acofactor, pursuant to the present invention.

FIGS. 2.3A2 to J2 show variations of the components of coferon drugdesign. FIG. 2.3A2 is a schematic drawing of two coferon monomers inaccordance with the present invention attached to an encoded bead viaseparate connectors. FIG. 2.3B2 is a schematic drawing of a tetheredcoferon dimer in accordance with the present invention with a DNAbarcode attached through a connector. FIG. 2.3C is a schematic drawingof a tethered coferon dimer attached to an encoded bead via a connectorto the linker elements. FIG. 2.3D2 is a schematic drawing of a tetheredcoferon dimer, in accordance with the present invention. FIG. 2.3E2 is aschematic drawing of a coferon trimer consisting of two coferon monomersattached to an encoded bead via connectors and a coferon monomer with aDNA barcode attached through a connector. The linker elements of thelatter monomer and one of the former monomers are joined. FIG. 2.3F2 isa schematic drawing of a coferon trimer including two tethered coferonmonomers with a DNA barcode attached via connectors and a coferonmonomer with a DNA barcode attached through a connector. The linkerelements of the latter monomer and one of the former monomers arejoined. FIG. 2.3G2 is a schematic drawing of a coferon trimer includingtwo coferons with the same ligand, and one coferon with a differentligand, pursuant to the present invention. The linker elements of thelatter monomer and one of the former monomers are joined. FIG. 4H2 is aschematic drawing of a coferon trimer including two coferon monomers inaccordance with the present invention attached to an encoded bead viathe linker element, and another coferon monomer. The linker elements ofthe latter monomer and one of the former monomers are joined. FIG. 2.3I2is a schematic drawing of a coferon trimer comprising a coferon monomerattached to an encoded bead through a connector, and two other coferonmonomers, each with a DNA barcode attached through a connector. Thelinker elements of the former monomer and one of the latter monomers arejoined. FIG. 2.3J2 is a schematic drawing of a coferon trimer comprisingthree monomer coferons with different ligands, pursuant to the presentinvention.

FIGS. 2.4K2 to T2 show variations of the components of coferon drugdesign. FIG. 2.4K2 is a schematic drawing of two coferon monomers inaccordance with the present invention attached to an encoded bead vialinker elements. FIG. 2.4L2 is a schematic drawing of a tethered coferondimer in accordance with the present invention with a DNA barcodeattached through a linker element. FIG. 2.4M2 is a schematic drawing ofa tethered coferon dimer attached to an encoded bead via a tether to thelinker elements. FIG. 2.4N2 is a schematic drawing of a tethered coferondimer, in accordance with the present invention. FIG. 2.4O2 is aschematic drawing of a coferon trimer including two coferon monomersattached to an encoded bead via their linker element and a coferonmonomer with a DNA barcode attached through a linker element. FIG. 2.4P2is a schematic drawing of a coferon trimer comprising two tetheredcoferon monomers with a DNA barcode attached via a linker element and acoferon monomer with a DNA barcode attached through a linker element.FIG. 2.4Q2 is a schematic drawing of a coferon trimer including twocoferons with the same ligand, and one coferon with a different ligand,pursuant to the present invention. FIG. 5R2 is a schematic drawing of acoferon trimer including two tethered coferon monomers in accordancewith the present invention attached to an encoded bead via theirtethered linker elements, and another coferon monomer which is joinedthrough to one of the tethered monomers by their linker elements. FIG.5S2 is a schematic drawing of a coferon trimer comprising a coferonmonomer attached to an encoded bead through a linker element, and twoother coferon monomers, each with a DNA barcode attached through aconnector. FIG. 2.4T2 is a schematic drawing of a coferon trimerincluding three monomer coferons with different ligands, pursuant to thepresent invention.

FIGS. 2.5A3 to L3 show the variations of the components of coferon drugdesign. FIG. 2.5A3 is a schematic drawing of a coferon tetramercomprised of two coferon monomers in accordance with the presentinvention attached to an encoded bead via connectors and to themselvesvia their linker element, and a tethered coferon dimer in accordancewith the present invention with a DNA barcode attached through aconnector. FIG. 2.5B3 is a schematic drawing of a coferon tetramercomprised of a tethered coferon dimer in accordance with the presentinvention with a DNA barcode attached through a connector, and anothertethered coferon dimer in accordance with the present invention with aDNA barcode attached through a connector. FIG. 2.5C3 is a schematicdrawing of a coferon tetramer including two monomer coferons with oneligand and two monomer coferons with another ligand pursuant to thepresent invention. FIG. 2.5D3 is a schematic drawing of a coferontetramer comprised of a tethered coferon dimer in accordance with thepresent invention attached to an encoded bead via tethers, and atethered coferon dimer in accordance with the present invention with aDNA barcode attached through a connector. FIG. 2.5E3 is a schematicdrawing of a coferon tetramer comprised of a tethered coferon dimer inaccordance with the present invention attached to an encoded bead viatethers, and a tethered coferon dimer in accordance with the presentinvention. FIG. 2.5F3 is a schematic drawing of a coferon tetramercomprised of two coferon monomers in accordance with the presentinvention attached to an encoded bead via connectors, and a tetheredcoferon dimer in accordance with the present invention. FIG. 2.5G3 is aschematic drawing of a coferon tetramer comprised of two coferonmonomers in accordance with the present invention attached to an encodedbead via connectors, and two coferon monomers with different ligands inaccordance with the present invention with DNA barcodes attached throughconnectors. FIG. 2.5H3 is a schematic drawing of a coferon tetramercomprised of a tethered coferon dimer in accordance with the presentinvention with a DNA barcode attached through a connector, and twotethered coferon monomers with different ligands in accordance with thepresent invention with DNA barcodes attached through connectors. FIG.2.5I3 is a schematic drawing of a coferon tetramer consisting of twomonomer coferons with one ligand and two monomer coferons with twodifferent ligands pursuant to the present invention. FIG. 2.5J3 is aschematic drawing of a coferon tetramer comprised of a coferon dimer inaccordance with the present invention attached to an encoded bead viathe tether, and two coferon monomers with different ligands inaccordance with the present invention with DNA barcodes attached throughconnectors. FIG. 2.5K3 is a schematic drawing of a coferon tetramercomprised of a tethered coferon dimer in accordance with the presentinvention attached to encoded beads via the tether, and two coferonmonomers with different ligands in accordance with the presentinvention. FIG. 2.5L3 is a schematic drawing of a coferon tetramercomprised of two coferon monomers in accordance with the presentinvention attached to an encoded bead via connector, and two coferonmonomers with different ligands in accordance with the presentinvention.

FIGS. 2.6M3-X3 show variations of the components of coferon drug design.FIG. 2.6M3 is a schematic drawing of a coferon tetramer comprised of twocoferon monomers in accordance with the present invention attached to anencoded bead via linker elements, and a tethered coferon dimer inaccordance with the present invention with a DNA barcode attachedthrough a linker element. FIG. 2.6N3 is a schematic drawing of a coferontetramer comprised of a tethered coferon dimer in accordance with thepresent invention with a DNA barcode attached through a linker element,and another tethered coferon dimer in accordance with the presentinvention with a DNA barcode attached through a linker element. FIG.2.6O3 is a schematic drawing of a coferon tetramer comprising twomonomer coferons with one ligand and two monomer coferons with anotherligand pursuant to the present invention. FIG. 2.6P3 is a schematicdrawing of a coferon tetramer comprised of a tethered coferon dimer inaccordance with the present invention attached to an encoded bead viatethers, and a tethered coferon dimer in accordance with the presentinvention with a DNA barcode attached through a linker element. FIG.2.6Q3 is a schematic drawing of a coferon tetramer comprised of atethered coferon dimer in accordance with the present invention attachedto an encoded bead via tethers, and a tethered coferon dimer inaccordance with the present invention. FIG. 2.6R3 is a schematic drawingof a coferon tetramer comprised of two tethered coferon dimers inaccordance with the present invention attached to an encoded bead vialinker elements in accordance with the present invention. FIG. 2.6S3 isa schematic drawing of a coferon tetramer comprised of two coferonmonomers in accordance with the present invention attached to an encodedbead via linker elements, and two coferon monomers with differentligands in accordance with the present invention with DNA barcodesattached through linker elements. FIG. 2.6T3 is a schematic drawing of acoferon tetramer comprised of a tethered coferon dimer in accordancewith the present invention with DNA barcodes attached through the linkerelements, and two coferon monomers with different ligands in accordancewith the present invention with DNA barcodes attached through linkerelements. FIG. 2.6U3 is a schematic drawing of a coferon tetramerconsisting of two coferon monomers with one ligand and two coferonmonomers with two different ligands pursuant to the present invention.FIG. 2.6V3 is a schematic drawing of a coferon tetramer comprised of atethered coferon dimer in accordance with the present invention attachedto an encoded bead via the tether, and two coferon monomers withdifferent ligands in accordance with the present invention with DNAbarcodes attached through linker elements. FIG. 2.6W3 is a schematicdrawing of a coferon tetramer comprised of a tethered coferon dimer inaccordance with the present invention attached to an encoded bead viathe tether, and two coferon monomers with different ligands inaccordance with the present invention. FIG. 2.6X3 is a schematic drawingof a coferon tetramer comprised of two coferon monomers in accordancewith the present invention attached to an encoded bead via linkerelements, and two coferon monomers with different ligands in accordancewith the present invention.

FIGS. 2.7A4-F4 show variations of the components of coferon drug design.FIG. 2.7A4 is a schematic drawing of a coferon tetramer comprised offour tethered coferon monomers with the same linker elements,connectors, and the same ligands in accordance with the presentinvention attached to an encoded bead via tethers. FIG. 2.7B4 is aschematic drawing of a coferon tetramer comprised of four tetheredcoferon monomers with the same linker elements, connectors, and the sameligands in accordance with the present invention with a DNA barcodeattached through the tether. FIG. 2.7C4 is a schematic drawing of acoferon tetramer comprised of four tethered coferon monomers with twodifferent linker elements, connectors, and the same ligands inaccordance with the present invention attached to encoded beads viatethers. FIG. 2.7D4 is a schematic drawing of a coferon tetramercomprised of four tethered coferon monomers with two different linkerelements, connectors, and the same ligands in accordance with thepresent invention with a DNA barcode attached through the tether. FIG.2.7E4 is a schematic drawing of a coferon tetramer comprised of fourtethered coferon monomers with two different linker elements,connectors, and two different ligands in accordance with the presentinvention attached to an encoded bead via tethers. FIG. 2.7F4 is aschematic drawing of a coferon tetramer comprised of four tetheredcoferon monomers with two different linker elements, connectors, and twodifferent ligands in accordance with the present invention with a DNAbarcode attached through the tether.

FIGS. 2.8G4-N4 show variations of the components of coferon drug design.FIG. 2.8G4 is a schematic drawing of a coferon dimer comprised of twotethered coferon monomers with the same linker elements, connectors, andthe same ligands in accordance with the present invention attached to anencoded bead via tethers. FIG. 2.8H4 is a schematic drawing of a coferondimer comprised of two tethered coferon monomers with the same linkerelements, connectors, and the same ligands in accordance with thepresent invention with a DNA barcode attached through the tether. FIG.2.8I4 is a schematic drawing of a coferon tetramer comprised of twotethered coferon monomers with the same ligands in accordance with thepresent invention attached to an encoded bead via tethers and thecoferon monomers comprised of two tethered coferon monomers with thesame ligands in accordance with the present invention with a DNA barcodeattached through the tether. FIG. 2.8 J4 is a schematic drawing of acoferon tetramer comprised of two tethered coferon monomers with thesame ligands in accordance with the present invention attached to anencoded bead via tethers and a coferon dimer comprised of two tetheredcoferon monomers with the same ligands in accordance with the presentinvention. FIG. 2.8K4 is a schematic drawing of a coferon tetramerconsisting of two coferon monomers with one ligand and two coferonmonomers with another ligand pursuant to the present invention. FIG.2.8L4 is a schematic drawing of a coferon tetramer comprised of twotethered coferon monomers with the same ligands in accordance with thepresent invention attached to an encoded bead via tethers and twocoferon monomers with two different ligands in accordance with thepresent invention with DNA barcodes attached through the linkerelements. FIG. 2.8M4 is a schematic drawing of a coferon tetramercomprised of two tethered coferon monomers with the same ligands inaccordance with the present invention attached to an encoded bead viatethers and two coferon monomers with two different ligands inaccordance with the present invention. FIG. 2.8N4 is a schematic drawingof a coferon tetramer consisting of two coferon monomers with one ligandand two coferon monomers with two different ligands pursuant to thepresent invention.

FIGS. 2.9O4-T4 show variations of the components of coferon drug design.FIG. 2.9O4 is a schematic drawing of a coferon tetramer comprising fourmonomer coferons with the same ligand pursuant to the present invention.FIG. 2.9P4 is a schematic drawing of a coferon tetramer including fourmonomer coferons with two different linker elements but the same ligandpursuant to the present invention. FIG. 12.9Q4 is a schematic drawing ofa coferon tetramer comprising two coferon monomers with one ligand andtwo coferon monomers with another ligand pursuant to the presentinvention. FIG. 2.9R4 is a schematic drawing of a coferon dimerincluding of two monomer coferons with the same ligand pursuant to thepresent invention. FIG. 2.9S4 is a schematic drawing of a coferon dimerconsisting of two coferon monomers with two different linker elementsbut the same ligand pursuant to the present invention. FIG. 2.9T4 is aschematic drawing of a coferon dimer consisting of one coferon monomerwith one ligand and one coferon monomer with another ligand pursuant tothe present invention.

FIGS. 2.10A5-F5 show variations of the components of coferon drugdesign. FIG. 2.10A5 is a schematic drawing of a coferon tetramercomprised of four tethered coferon monomers with the same linkerelements and the same ligands in accordance with the present inventionattached to an encoded bead via tethers. FIG. 2.10B5 is a schematicdrawing of a coferon tetramer comprised of four tethered coferonmonomers with the same linker elements and the same ligands inaccordance with the present invention with a DNA barcode attachedthrough the tether. FIG. 2.10C5 is a schematic drawing of a coferontetramer comprised of four tethered coferon monomers with two differentlinker elements and the same ligands in accordance with the presentinvention attached to an encoded bead via tethers. FIG. 2.10B5 is aschematic drawing of a coferon tetramer comprised of four tetheredcoferon monomers with two different linker elements and the same ligandsin accordance with the present invention with a DNA barcode attachedthrough the tether. FIG. 2.10E5 is a schematic drawing of a coferontetramer comprised of four tethered coferon monomers with two differentlinker elements and two different ligands in accordance with the presentinvention attached to encoded beads via tethers. FIG. 2.10F5 is aschematic drawing of a coferon tetramer comprised of four tetheredcoferon monomers with two different linker elements and two differentligands in accordance with the present invention with a DNA barcodeattached through the tether.

FIGS. 2.11G5-N5 show the variations of the components of coferon drugdesign. FIG. 2.11G5 is a schematic drawing of a coferon dimer comprisedof two tethered coferon monomers with the same linker elements and thesame ligands in accordance with the present invention attached to anencoded bead via tethers. FIG. 2.11H5 is a schematic drawing of acoferon dimer comprised of two tethered coferon monomers with the samelinker elements and the same ligands in accordance with the presentinvention with a DNA barcode attached through the tether. FIG. 2.11I5 isa schematic drawing of a coferon tetramer comprised of two tetheredcoferon monomers with the same ligands in accordance with the presentinvention attached to an encoded bead via tethers and a coferon dimercomprised of two tethered coferon monomers with the same ligands inaccordance with the present invention with a DNA barcode attachedthrough the tether. FIG. 2.11J5 is a schematic drawing of a coferontetramer comprised of two tethered coferon monomers with the sameligands in accordance with the present invention attached to an encodedbead via tethers and a coferon dimer comprised of two tethered coferonmonomers with the same ligands in accordance with the present invention.FIG. 2.11K5 is a schematic drawing of a coferon tetramer consisting oftwo coferon monomers with one ligand and two coferon monomers withanother ligand pursuant to the present invention. FIG. 2.11L5 is aschematic drawing of a coferon tetramer comprised of two tetheredcoferon monomers with the same ligands in accordance with the presentinvention attached to an encoded bead via tethers and two coferonmonomers with two different ligands in accordance with the presentinvention with DNA barcodes attached through the linker elements. FIG.2.11M5 is a schematic drawing of a coferon tetramer comprised of twotethered coferon monomers with the same ligands in accordance with thepresent invention attached to encoded beads via tethers and two coferonmonomers with two different ligands in accordance with the presentinvention. FIG. 2.11N5 is a schematic drawing of a coferon tetramerconsisting of two monomer coferons with one ligand and two monomercoferons with two different ligands pursuant to the present invention.

FIGS. 2.12O5-T5 show variations of the components of coferon drugdesign. FIG. 2.12O5 is a schematic drawing of a coferon tetramerincluding four monomer coferons with the same ligand pursuant to thepresent invention. FIG. 2.12P5 is a schematic drawing of a coferontetramer comprising four monomer coferons with two different linkerelements but the same ligand pursuant to the present invention. FIG.2.12Q5 is a schematic drawing of a coferon tetramer including twomonomer coferons with one ligand and two monomer coferons with anotherligand pursuant to the present invention. FIG. 2.12R5 is a schematicdrawing of a coferon dimer comprising two coferon monomers with the sameligand pursuant to the present invention. FIG. 2.12S5 is a schematicdrawing of a coferon dimer including two coferon monomers with twodifferent linker elements but the same ligand pursuant to the presentinvention. FIG. 2.12T5 is a schematic drawing of a coferon dimercomprising one coferon monomer with one ligand and one coferon monomerwith another ligand pursuant to the present invention.

FIGS. 2.13A6-F6 show variations of the components of coferon drugdesign. FIG. 2.13A6 is a schematic drawing of a coferon hexamercomprised of six tethered coferon monomers with the same linkerelements, connectors, and the same ligands in accordance with thepresent invention attached to an encoded bead via tethers. FIG. 2.13B6is a schematic drawing of a coferon hexamer comprised of six tetheredcoferon monomers with the same linker elements, connectors, and the sameligands in accordance with the present invention with a DNA barcodeattached through the tether. FIG. 2.13C6 is a schematic drawing of acoferon hexamer comprised of six tethered coferon monomers with twodifferent linker elements, connectors, and the same ligands inaccordance with the present invention attached to an encoded bead viatethers. FIG. 2.13D6 is a schematic drawing of a coferon hexamercomprised of six tethered coferon monomers with two different linkerelements, connectors, and the same ligands in accordance with thepresent invention with a DNA barcode attached through the tether. FIG.2.13E6 is a schematic drawing of a coferon hexamer comprised of sixtethered coferon monomers with two different linker elements,connectors, and two different ligands in accordance with the presentinvention attached to encoded beads via tethers. FIG. 2.13F6 is aschematic drawing of a coferon hexamer comprised of six tethered coferonmonomers with two different linker elements, connectors, and twodifferent ligands in accordance with the present invention with a DNAbarcode attached through the tether.

FIGS. 2.14G6-N6 show variations of the components of coferon drugdesign. FIG. 2.14G6 is a schematic drawing of a coferon trimer comprisedof three tethered coferon monomers with the same linker elements,connectors, and the same ligands in accordance with the presentinvention attached to an encoded bead via tethers. FIG. 2.14H6 is aschematic drawing of a coferon trimer comprised of three tetheredcoferon monomers with the same linker elements, connectors, and the sameligands in accordance with the present invention with a DNA barcodeattached through the tether. FIG. 2.14I6 is a schematic drawing of acoferon hexamer comprised of three tethered coferon monomers with thesame ligands in accordance with the present invention attached toencoded beads via tethers and a coferon trimer comprised of threetethered coferon monomers with the same ligands in accordance with thepresent invention with a DNA barcode attached through the tether. FIG.2.14J6 is a schematic drawing of a coferon hexamer comprised of threetethered coferon monomers with the same ligands in accordance with thepresent invention attached to an encoded bead via tethers and a coferontrimer comprised of three tethered coferon monomers with the sameligands in accordance with the present invention. FIG. 2.14K6 is aschematic drawing of a coferon hexamer including three coferon monomerswith one ligand and three coferon monomers with another ligand pursuantto the present invention. FIG. 2.14L6 is a schematic drawing of acoferon hexamer comprising six coferon monomers with the same ligandpursuant to the present invention. FIG. 2.14M6 is a schematic drawing ofa coferon hexamer including six coferon monomers with two differentlinker elements but the same ligand pursuant to the present invention.FIG. 2.14N6 is a schematic drawing of a coferon trimer comprising twocoferon monomers with one linker element and the same ligand, and onecoferon monomer with another linker element and another ligand pursuantto the present invention.

FIGS. 2.15A7-F7 show variations of the components of coferon drugdesign. FIG. 2.15A7 is a schematic drawing of a coferon hexamercomprised of six tethered coferon monomers with the same linker elementsand the same ligands in accordance with the present invention attachedto an encoded bead via tethers. FIG. 2.15B7 is a schematic drawing of acoferon hexamer comprised of six tethered coferon monomers with the samelinker elements, and the same ligands in accordance with the presentinvention with a DNA barcode attached through the tether. FIG. 2.15C7 isa schematic drawing of a coferon hexamer comprised of six tetheredcoferon monomers with two different linker elements and the same ligandsin accordance with the present invention attached to encoded beads viatethers. FIG. 2.15B7 is a schematic drawing of a coferon hexamercomprised of six tethered coferon monomers with two different linkerelements and the same ligands in accordance with the present inventionwith a DNA barcode attached through the tether. FIG. 2.15E7 is aschematic drawing of a coferon hexamer comprised of six tethered coferonmonomers with two different linker elements and two different ligands inaccordance with the present invention attached to an encoded beadtethers. FIG. 2.15F7 is a schematic drawing of a coferon hexamercomprised of six tethered coferon monomers with two different linkerelements and two different ligands in accordance with the presentinvention with a DNA barcode attached through the tether.

FIGS. 2.16G7-N7 show variations of the components of coferon drugdesign. FIG. 2.16G7 is a schematic drawing of a coferon trimer comprisedof three tethered coferon monomers with the same linker elements and thesame ligands in accordance with the present invention attached to anencoded bead via tethers. FIG. 2.16H7 is a schematic drawing of acoferon trimer comprised of three tethered coferon monomers with thesame linker elements and the same ligands in accordance with the presentinvention with a DNA barcode attached through the tether. FIG. 2.16I7 isa schematic drawing of a coferon hexamer comprised of three tetheredcoferon monomers with the same ligands in accordance with the presentinvention attached to encoded beads via tethers and a coferon trimercomprised of three tethered coferon monomers with the same ligands inaccordance with the present invention with a DNA barcode attachedthrough the tether. FIG. 2.16J7 is a schematic drawing of a coferonhexamer comprised of three tethered coferon monomers with the sameligands in accordance with the present invention attached to an encodedbead via tethers and a coferon trimer comprised of three tetheredcoferon monomers with the same ligands in accordance with the presentinvention. FIG. 2.16K7 is a schematic drawing of a coferon hexamerincluding three coferon monomers with one ligand and three coferonmonomers with another ligand pursuant to the present invention. FIG.2.16L7 is a schematic drawing of a coferon hexamer comprising sixcoferon monomers with the same ligand pursuant to the present invention.FIG. 2.16M7 is a schematic drawing of a coferon hexamer including sixcoferon monomers with two different linker elements but the same ligandpursuant to the present invention. FIG. 2.16N7 is a schematic drawing ofa coferon trimer comprising two coferon monomers with one linker elementand the same ligand, and one coferon monomer with another linker elementand another ligand pursuant to the present invention.

FIGS. 2.17A-C show variations of coferon drug interactions with atarget. Coferon 1 is illustrated as a purple “L” linker element tetheredto a yellow hexagon ligand, coferon 2 as an orange “upsidedown L” linkerelement tethered to a green oval ligand, and the target protein as alight blue shape. A substrate is illustrated as a deep orange dumbbellshaped object, and the cleavage products as the two halves. A bindingpartner of the target is illustrated as a dark blue shape. FIG. 2.17A isa schematic drawing of a substrate binding to and being cleaved by thetarget. FIG. 2.17B is a schematic drawing of two coferon monomersbinding to and forming a coferon dimer on the target whose dissociationconstant is less than or equal to the dissociation constant of thesubstrate, thus inhibiting the substrate from binding to and beingcleaved by the target. FIG. 2.17C is a schematic drawing of two coferonmonomers binding to and forming a coferon dimer on the target whosedissociation constant is less than or equal to the dissociation constantof a binding protein, thus displacing the binding protein from bindingto the target.

FIGS. 2.18D-G show variations of coferon drug interactions with atarget. Coferon 1, coferon 2 and the target protein are described above.Activation of the target protein, for example, by turning on a kinaseactivity, is illustrated by an arc of red lines. An activating bindingpartner of the target is illustrated as a purple shape. An inhibitingbinding partner of the target is illustrated as a green shape. FIG.2.18D is a schematic drawing of an activating binding partner binding toand activating the target. FIG. 2.18E is a schematic drawing of twocoferon monomers binding to and forming a coferon dimer on the target,mimicking the activating binding partner by activating the target. FIG.2.18F is a schematic drawing of an inactivating binding partner bindingto and inactivating the target. FIG. 2.18G is a schematic drawing of twocoferon monomers binding to and forming a coferon dimer on the target,mimicking the inactivating binding partner by inactivating the target.

FIGS. 2.19H-I show the variations of coferon drug interactions with atarget. Coferon 1, coferon 2 and the target protein are described above.Activation of the target protein, for example, by turning on a kinaseactivity, is illustrated by an arc of red lines, with intensity ofactivation suggested by the number of red lines in the arc. Anactivating binding partner of the target is illustrated as a greenshape. An inhibiting binding partner of the target is illustrated as apurple shape. FIG. 2.19H is a schematic drawing of an activating bindingpartner binding to and mildly activating the target (upper pathway).Addition of two coferon monomers allows binding to and forming a coferondimer on the activating binding partner-target complex, thus enhancingactivation of the target (lower pathway). FIG. 2.19I is a schematicdrawing of an inactivating binding partner binding to and mildlyinactivating the target (upper pathway). Addition of two coferonmonomers allows binding to and forming a coferon dimer on the activatingbinding partner-target complex, thus enhancing inactivation of thetarget (lower pathway).

FIGS. 2.20J-K show variations of coferon drug interactions with atarget. Coferon 1, coferon 2 and the target protein are described above.A mutant target protein is illustrated as a light blue shape with a red“M”. Activation of the target protein, for example, by turning on akinase activity, is illustrated by an arc of red lines, with intensityof activation suggested by the number of red lines in the arc. Anactivating binding partner of the target is illustrated as a greenshape. FIG. 2.20J is a schematic drawing of an activating bindingpartner binding to and activating the wild-type target. FIG. 2.20J is aschematic drawing of an activating binding partner binding to and mildlyactivating the mutant target (upper pathway). Addition of two coferonmonomers allows binding to and forming a coferon dimer on the mutanttarget, thus enhancing activation of the mutant target (lower pathway).

FIGS. 2.21L-M show variations of coferon drug interactions with atarget. Coferon 1, coferon 2 and the target protein are described above.A mutant target protein is illustrated as a light blue shape with a red“M”. Inactivation of the target protein, is illustrated by (loss of) anarc of red lines, with intensity of activation suggested by the numberof red lines in the arc. An inactivating binding partner of the targetis illustrated as a purple shape. FIG. 2.21L is a schematic drawing ofan inactivating binding partner binding to and inactivating thewild-type target. FIG. 2.21M is a schematic drawing of an inactivatingbinding partner binding to and mildly inactivating the (overactivated)mutant target (upper pathway). Addition of two coferon monomers allowsbinding to and forming a coferon dimer on the mutant target, thusenhancing inactivation of the mutant target (lower pathway).

FIGS. 2.22N-O show variations of coferon drug interactions with atarget. Coferon 1, coferon 2 and the target protein are described above.A first binding partner, with weak affinity to the target is illustratedas a green shape. A second binding partner with affinity to the target,coferons, and first binding partner is illustrated as a deep orangeshape. FIG. 2.22N is a schematic drawing of the first binding partnerbinding weakly to the target. FIG. 2.22O is a schematic drawing of thefirst binding partner binding weakly to the target (upper pathway).Addition of two coferon monomers allows binding to and forming a coferondimer on the target, recruiting the second binding partner to bind tothe target, coferons, and first binding partner, forming a coferondimer-target-second binding protein complex, and thus enhancing bindingof the first binding partner to the target (lower pathway).

FIGS. 2.23P-Q show variations of coferon drug interactions with atarget. Coferon 1, coferon 2 and the target protein are described above.A first binding partner, with strong affinity to the target isillustrated as a dark blue shape. A second binding partner with affinityto the target and coferons is illustrated as a deep orange shape. FIG.2.23P is a schematic drawing of the first binding partner bindingstrongly to the target. FIG. 2.23Q is a schematic drawing of the firstbinding partner binding strongly to the target (upper pathway). Additionof two coferon monomers allows binding to and forming a coferon dimer onthe target, recruiting the second binding partner to bind to the targetand coferons forming a coferon dimer-target-second binding proteincomplex, whose dissociation constant is less than or equal to thedissociation constant of the first binding protein, thus displacing thefirst binding protein from binding to the target (lower pathway).

FIGS. 2.24R-T show the variations of coferon drug interactions with atarget. Coferon 1, coferon 2 and the target protein are described above.A first binding partner, with weak or no affinity to the target isillustrated as a green shape. A second binding partner with affinity tothe target, coferons, and/or first binding partner is illustrated as adeep orange shape. FIG. 2.24R is a schematic drawing of two coferonmonomers binding to and forming a coferon dimer on the target,recruiting the second binding partner to bind to the target, coferons,and first binding partner, forming a coferon dimer-target-second bindingprotein complex, and thus recruiting the first binding partner to thetarget. FIG. 2.24S is a schematic drawing of two coferon monomersbinding to and forming a coferon dimer on the target, recruiting thesecond binding partner to bind to the target, coferons, and firstbinding partner, forming a coferon dimer-target-second binding proteincomplex, and thus recruiting the first binding partner to the target.FIG. 2.24T is a schematic drawing of two coferon monomers binding to andforming a coferon dimer on the target and the first binding protein,recruiting the second binding partner to bind to the target and firstbinding partner, forming a coferon dimer-target-first bindingprotein-second binding protein complex, and thus recruiting the firstbinding partner to the target.

FIGS. 2.25A2-B2 show variations of coferon drug interactions with atarget. Coferon 1, coferon 2 and the target protein are described above.The natural ligand for the receptor dimer is a deep orange oval, and themembrane as a semi-transparent yellow line. Activation of the targetprotein, for example, by turning on a kinase activity, is illustrated byan arc of red lines, with intensity of activation suggested by thenumber of red lines in the arc. FIG. 2.25A2 is a schematic drawing of anactivating ligand binding to the receptor target, facilitating receptordimerization, and activating the receptor target. FIG. 2.25B2 is aschematic drawing of two coferon monomers binding to and forming acoferon dimer on the target, mimicking the activating ligand,facilitating receptor dimerization, and activating the receptor target.

FIGS. 2.26C2-D2 show variations of coferon drug interactions with atarget. Coferon 1, coferon 2 and the target protein are described above.The natural ligand for the receptor dimer is a deep orange oval, and themembrane is a semi-transparent yellow line. FIG. 2.26C2 is a schematicdrawing of two coferon monomers binding to and forming a coferon dimeron the target, interfering with proper receptor dimerization, andinhibiting activation of the receptor target. FIG. 2.26D2 is a schematicdrawing of two coferon monomers binding to and forming a coferon dimeron each target, inhibiting activation at an allosteric site, even in thepresence of activating ligand that facilitates receptor dimerization.

FIG. 2.27E2 shows variations of coferon drug interactions with a target.Coferon 1, coferon 2 and the target protein are described above. Thenatural ligand for the receptor dimer is a deep orange oval, and themembrane is a semi-transparent yellow line. Activation of the targetprotein, for example, by turning on a kinase activity, is illustrated byan arc of red lines, with intensity of activation suggested by thenumber of red lines in the arc. FIG. 2.27E2 is a schematic drawing oftwo coferon monomers binding to and forming a coferon dimer on eachtarget, enhancing activation at an allosteric site, which is enhanced inthe presence of activating ligand that facilitates receptordimerization.

FIGS. 2.28F2-H2 show variations of coferon drug interactions with atarget. Coferon 1, coferon 2 and the target protein are described above,The natural ligand for the receptor dimer is a deep orange imperfectoval, and the membrane is a semi-transparent yellow line. A bindingpartner, with affinity to the target upon binding its ligand, isillustrated as a green shape. Upon binding the target protein, thebinding partner may be activated, for example, by turning on a kinaseactivity, and is illustrated by an arc of red lines, with intensity ofactivation suggested by the number of red lines in the arc. FIG. 2.28F2is a schematic drawing of the natural ligand binding to the receptortarget, which recruits and activates the binding partner. FIG. 2.28G2 isa schematic drawing of two coferon monomers binding to and forming acoferon dimer on the receptor target at the ligand binding site to actas an agonist, mimicking the natural ligand, which recruits andactivates the binding partner. FIG. 2.28H2 is a schematic drawing of twocoferon monomers binding to and forming a coferon dimer on the receptortarget at the ligand binding site to act as an antagonist, and thusinhibits recruitment and activation of the binding partner.

FIGS. 2.29I2-K2 show variations of coferon drug interactions with atarget. Coferon 1, coferon 2, and the target protein are describedabove. The natural ligand for the receptor dimer is a deep orangeimperfect oval, and the membrane as a semi-transparent yellow line. Abinding partner, with affinity to the target upon binding its ligand, isillustrated as a green shape. Upon binding the target protein, thebinding partner may be activated, for example, by turning on a kinaseactivity, and is illustrated by an arc of red lines, with intensity ofactivation suggested by the number of red lines in the arc. FIG. 2.29I2is a schematic drawing of two coferon monomers binding to and forming acoferon dimer on the receptor target at the binding partner binding siteto act as an antagonist, and thus inhibit recruitment and activation ofthe binding partner. FIG. 2.29J2 is a schematic drawing of the naturalligand binding to the receptor target, which recruits and activates thebinding partner, with two coferon monomers binding to and forming acoferon dimer on the receptor target and the binding partner, to enhanceactivation of the binding partner. FIG. 2.29K2 is a schematic drawing ofthe natural ligand binding to the receptor target, which recruits andactivates the binding partner, with two coferon monomers binding to andforming a coferon dimer on the receptor target and the natural ligand,to enhance activation of the binding partner.

FIGS. 2.30A3-C3 show the variations of coferon drug interactions with atarget. Coferon 1 is illustrated as a purple cylindrical linker elementtethered to a yellow hexameric ligand, Coferon 2 is illustrated as anorange cylindrical linker element tethered to a green oval ligand, thedimer target protein as a light blue shape dimerized to the dark blueshape. FIG. 2.30A3 is a schematic drawing of two coferon monomersbinding to form a coferon homodimer on the dimer target. FIG. 2.30B3 isa schematic drawing of a coferon tetramer comprised of four coferonmonomers binding to form a coferon homotetramer on the dimer target.FIG. 2.30B3 is a schematic drawing of a coferon tetramer comprised oftwo coferon monomers with one ligand and two coferon monomers with asecond ligand binding to form a coferon heterotetramer on the dimertarget.

FIGS. 2.31D3-F3 show variations of coferon drug interactions with atarget. Coferon 1 is illustrated as a purple cylindrical linker elementtethered to a yellow hexameric ligand, Coferon 2 is illustrated as anorange cylindrical linker element tethered to a green oval ligand,Coferon 3 is illustrated as an light pink cylindrical linker elementtethered to a pink star ligand, the multimeric target proteins arecomprised of the larger cylinders with different shades of blue andorange. A cell membrane is illustrated as a semi-transparent yellowline. FIG. 2.31D3 is a schematic drawing of a coferon tetramer comprisedof two coferon monomers with one ligand and two coferon monomers with asecond ligand, binding to form a coferon heterotetramer on a multimerictarget. FIG. 2.31E3 is a schematic drawing of a coferon tetramercomprised of two coferon monomers with one ligand and two differentcoferon monomers with a second and third ligand, binding to form acoferon heterotetramer on a multimeric target. FIG. 2.31F3 is aschematic drawing of a coferon hexamer comprised of three coferonmonomers with one ligand and three coferon monomers with a secondligand, binding to form a coferon heterohexamer on a multimeric target.

FIGS. 2.32A4-B4 show the variations of coferon drug interactions with atarget. Coferon 1 is illustrated as a purple “L” linker element tetheredto a yellow hexameric ligand, Coferon 2 is illustrated as an orange “L”linker element tethered to a green oval ligand, and the target tubulinheterodimer as the blue and purple circles. FIG. 2.32A4 is a schematicdrawing of alpha and beta tubulin heterodimers combining to formpolymerized tubulin filaments. FIG. 2.32B4 is a schematic drawing of twocoferon monomers binding to form a coferon dimer on the tubulin dimertarget, thus destabilizing filament formation.

FIGS. 2.33D4-E4 show variations of coferon drug interactions with atarget. Coferon 1 is illustrated as a purple “L” linker element tetheredto a yellow hexameric ligand, Coferon 2 is illustrated as an orange “L”linker element tethered to a green oval ligand, the target amyloid betapeptide as the blue hexamers, circles, and rounded squares. FIG. 2.33D4is a schematic drawing of amyloid beta peptide monomers aggregating toform small oligomers, large oligomers, protofibriles and amyloid fibrilsthat cause Alzheimer's Disease. FIG. 2.33E4 is a schematic drawing oftwo coferon monomers binding to form a coferon dimer on the amyloid betapeptide monomers, thus inhibiting aggregation and disease.

FIGS. 2.34A-B show variations of coferon drug interactions with an RNAtarget. Coferon 1 is illustrated as a purple “L” linker element tetheredto a thick blue line illustrating RNA or oligomer nucleotide analoguethat is complementary to the desired target, Coferon 2 is illustrated asan orange “L” linker element tethered to a thick green line illustratingRNA or oligomer nucleotide analogue that is complementary to the desiredtarget, the desired RNA target illustrated as a deep orange line withcomplementary sequences illustrated as light blue and light green lines,with the incorrect RNA targets illustrated as light orange line withcomplementary or nearly complementary sequences illustrated as lightblue and light green lines, and with the membrane illustrated as a thickcurved line. FIG. 2.34A is a schematic drawing of the antisenseoligonucleotide, which is transported across the membrane, to bind thedesired RNA target, obtaining the desired biological effect, as well assome undesired off-target effects. FIG. 2.34B is a schematic drawing ofthe coferon oligonucleotides, which are transported across the membrane,they can bind adjacent to each other on the desired RNA target bringingthe linker elements into proximity and allowing for formation of eitherreversible or irreversible bonds—greatly lowering the dissociationconstant. If the coferon oligonucleotide monomers bind on off targetsites, they will dissociate. Thus the coferon design allows forantisense oligonucleotide therapeutics to obtain the desired biologicaleffect of inhibiting a specific RNA target, while minimizing undesiredoff-target effects.

FIG. 2.35C shows variations of coferon drug interactions with an RNAtarget. Coferon 1 is illustrated as a purple “L” linker element tetheredto a thick blue line illustrating RNA or oligomer nucleotide analoguethat is complementary to the desired target, Coferon 2 is illustrated asan orange “L” linker element tethered to a thick green line illustratingRNA or oligomer nucleotide analogue that is complementary to the desiredtarget, lipocoferons or aminoglycoferons illustrated as short lightpurple bars tethered by reversible linkers such as disulfide bonds, thedesired RNA target illustrated as a deep orange line with complementarysequences illustrated as light blue and light green lines, with theincorrect RNA targets illustrated as light orange line withcomplementary or nearly complementary sequences illustrated as lightblue and light green lines, and with the membrane illustrated as a thickcurved line. FIG. 22.35C is a schematic drawing of the coferonoligonucleotides bound to lipocoferons or aminoglycoferons to formlipophilic complexes, which are transported across the membrane. Onceinside, intracellular glutathione reduces the disulfide bonds in thelipocoferons or aminoglycoferons, and they dissociate from the coferonoligonucleotides. The coferon oligonucleotides can then bind adjacent toeach other on the desired RNA target bringing the linker elements intoproximity and allowing for formation of either reversible orirreversible bonds—greatly lowering the dissociation constant. If thecoferon oligonucleotide monomers bind on off target sites, they willdissociate. Thus, the coferon design allows for antisenseoligonucleotide therapeutics to obtain the desired biological effect ofinhibiting a specific RNA target, while minimizing undesired off-targeteffects.

FIG. 2.36D shows variations of coferon drug interactions with an RNAtarget. Coferon 1 is illustrated as a purple “L” linker element tetheredto a thick blue line above an offset thick light blue line illustratingdouble-stranded RNA or oligomer nucleotide analogue that iscomplementary to the desired target, Coferon 2 is illustrated as anorange “L” linker element tethered to a thick green line above an offsetthick light green line illustrating double-stranded RNA or oligomernucleotide analogue that is complementary to the desired target,lipocoferons or aminoglycoferons illustrated as short light purple barstethered by reversible linkers such as disulfide bonds. The RISC complexis illustrated as a light gray rounded rectangle, the desired RNA targetillustrated as a deep orange line with complementary sequencesillustrated as light blue and light green lines, with the incorrect RNAtargets illustrated as light orange line with complementary or nearlycomplementary sequences illustrated as light blue and light green lines,and with the membrane illustrated as a thick curved line. FIG. 2.36D isa schematic drawing of the coferon oligonucleotides bound tolipocoferons or aminoglycoferons to form lipophilic complexes, which aretransported across the membrane. Once inside, intracellular glutathionereduces the disulfide bonds in the lipocoferons or aminoglycoferons, andthey dissociate from the coferon oligonucleotides. The coferonoligonucleotides can then bind adjacent to each other within the RISCcomplex, bringing the linker elements into proximity and allowing forformation of either reversible or irreversible bonds—and allowing forthe complementary strands to be destroyed. The coferon dimer within theRISC complex can now enzymatically degrade multiple copies of thedesired RNA target.

FIG. 3 shows a variation of encoding beads with unique identifiersequences for coferon ligand diversity synthesis. DNA with universalsequences is illustrated as a black line, while DNA with uniquesequences is illustrated as a colored line. A bead is illustrated as anarc. Coferon 1 is illustrated as an orange “L” linker element tetheredto a yellow hexagon ligand, coferon 2 as an orange “L” linker elementtethered to a pink star ligand, and coferon 3 as an orange “L” linkerelement tethered to a green oval ligand. Two sets of oligonucleotideprimers containing universal sequences at their ends and uniquesequences in the middle are used to generate PCR products of the form:UniA (20)-Rand (20)-UniB (15)-Rand (20)-UniC (20), where UniA isuniversal sequence A, UniB is universal sequence B, UniC is universalsequence C, and Rand designates random (unique) sequences of about 20bases in length. These are then amplified on unique beads using emulsionPCR, where one of the universal primers is on the beads. Coferons may besynthesized on activated groups directly on the surface of the bead, orin a variation, a primer containing a functional group is ligated to theend of the amplified product. Thus, the coferon will be synthesized atthe end of the DNA strand. This presents two opportunities: (a) with thecoferons at the end of the DNA, they are in easy reach of the proteintarget, and (b) this design provides the option of either cleaving thecoferon off the end of the bead, or cleaving the DNA off the bead, andthen it is still attached to the coferon.

FIG. 4 shows a variation of encoding beads with unique identifiersequences for coferon ligand diversity synthesis. DNA with universalsequences is illustrated as a black line, while DNA with uniquesequences is illustrated as a colored line. A bead is illustrated as arounded square. Coferon 1 is illustrated as an orange “L” linker elementtethered to a yellow hexagon ligand, coferon 2 as an orange “L” linkerelement tethered to a pink star ligand, and coferon 3 as an orange “L”linker element tethered to a green oval ligand. Two sets ofoligonucleotide primers containing universal sequences at their ends andunique sequences in the middle are used to generate PCR products of theform: UniA (20)-Rand (20)-UniB (15)-Rand (20)-UniC (20), where UniA isuniversal sequence A, UniB is universal sequence B, UniC is universalsequence C, and Rand designates random (unique) sequences of about 20bases in length. The beads are then activated on only one side. One mayfloat the beads on the surface of a liquid, and spraying an activatingchemical on only the exposed surface. Another approach is to print theactivating group on the surface of a silicon or glass wafer, and etchaway horizontal and vertical sections, leaving millions of particlesthat are just etched on one side. The random primers are then amplifiedon only one side of unique beads using emulsion PCR, where one of theuniversal primers is on the beads. Coferons may be synthesized onactivated groups directly on the other surfaces of the bead. Theadvantage of limiting the PCR product to one face of the bead is toavoid having too many DNA molecules sticking out, that may laterinterfere with the coferon synthesis or binding of fluorescently labeledtarget (protein) to the bead.

FIG. 5 shows a variation of encoding beads with unique identifiersequences for coferon ligand diversity synthesis. DNA with universalsequences is illustrated as a black line, the UniD sequence is indicatedby a blue line, while DNA with unique sequences is illustrated as acolored line. A bead is illustrated as a rounded square. Coferon 1 isillustrated as an orange “L” linker element tethered to a yellow hexagonligand, coferon 2 as an orange “L” linker element tethered to a pinkstar ligand, and coferon 3 as an orange “L” linker element tethered to agreen oval ligand. Two sets of oligonucleotide primers containinguniversal sequences at their ends and unique sequences in the middle areused to generate rolling circle products of the form: UniA (20)-Rand(20)-UniB (15)-Rand (20)-UniC (20)-UniD (20), where UniA is universalsequence A, UniB is universal sequence B, UniC is universal sequence C,UniD is universal sequence D, and Rand designates random (unique)sequences of about 20 bases in length. The beads are then activated ononly one side. One may float the beads on the surface of a liquid, andspraying an activating chemical on only the exposed surface. Anotherapproach is to print the activating group on the surface of a silicon orglass wafer, and etch away horizontal and vertical sections, leavingmillions of particles that are just etched on one side. The rollingcircle amplicons are then captured on only one side of unique beadsusing the complementary sequence of the UniD primer on the beads.Coferons may be synthesized on activated groups directly on the othersurfaces of the bead. The advantage of limiting the rolling circleproduct to one face of the bead is to avoid having too many DNAmolecules sticking out, that may later interfere with the coferonsynthesis or binding of fluorescently labeled target (protein) to thebead.

FIG. 6 shows a space filling 3-dimensional representation of linkerelements derived from 2-hydroxycyclohexanone.

FIG. 7 shows a space filling 3-dimensional representation of the dimerformed by linker elements derived from 2-hydroxycyclohexanone.

FIG. 8 shows a space filling 3-dimensional representation of the dimerformed by linker elements derived from 1-hydroxy-2-hydroxymethylcyclopentanecarbaldehyde.

FIG. 9 shows a space filling 3-dimensional representation of triazolelinked aromatic linker elements.

FIG. 10 shows a 3-dimensional representation of triazole linked elementsdimerized through intercalation. The translucent surface represents asolvent accessible area.

FIG. 11 shows a 3-dimensional representation of aromatic intercalatorbased linker element monomer.

FIG. 12 shows a side view of a 3-dimensional representation of a linkerelement dimer with molecular surfaces.

FIG. 13 is a top view of a 3-dimensional representation of a linkerelement dimer with molecular surfaces.

FIGS. 14A-B are schematic drawings for linker element screening inaccordance with the present invention.

FIGS. 15A-B are schematic drawings for linker element screening inaccordance with the present invention.

FIGS. 16A-C are schematic drawings of components used in diversityelement library synthesis for DNA encoded libraries. FIG. 16A shows DNAtemplate synthesis. FIG. 16B shows DNA sorted synthesis. FIG. 16C showszipcode capture synthesis.

FIGS. 17A-C are schematic drawings of components used in diversityelement library synthesis for bead encoded libraries. FIG. 17A showssmall molecule inhibitors and analogues. FIGS. 17B-17C showcombinatorial chemistry on a common platform.

FIG. 18 A-C are schematic drawings of directed evolution of coferons.

FIG. 19 is a schematic overview of selection of the tightest bindingcoferon protein interaction dimers taking advantage of the principles ofdynamic combinatorial chemistry. As shown in step 1, each coferonmomoner comprises a low MW binding ligand (diversity element) covalentlylinked to an encoding DNA strand (allowing for stepwise synthesis andidentification of the binding ligand) as well as a low MW linker element(dynamic combinatorial chemistry element). Under physiologicalconditions, as shown in step 2, different combinations of ligands areforming and reassociating with each other. When the diversity elementsare brought in contact with the protein target on a surface, as shown instep 3, some combinations will bind tighter than others. This directsthe evolution of combinations to the preferred pairs. After removingunbound ligands, DNA encoding regions (colored lines, “zip-codes”) maybe amplified using universal primers (black lines) to identifyindividual ligands, which serve as lead molecules.

FIG. 20 is a first embodiment for target screening in accordance withthe present invention. In step 1, a library of ligands is synthesized onbeads which may be individually identified through barcodes. Eachmonomer element consists of a low MW (approx. 600-800) binding ligands(diversity element) covalently linked to a low MW (approx. under 300)linker element (dynamic combinatorial chemistry element). The beads areincubated with fluorescently labeled target protein to identify ligandsthat bind most tightly to the target. In step 2, the top set of theseligands are resynthesized with the option of adding additional diversityin the connector between the linker element and the ligand diversityelement. A second library of ligands is synthesized, where each monomerelement consists of a low MW binding ligand (diversity element)covalently linked to a DNA barcode (allowing for stepwise synthesis andidentification of the binding ligand) as well as a low MW linkerelement, suitable for reversible binding to the linker element elementin the first library. Under physiological conditions, differentcombinations of ligands are forming and reassociating with each other.The surface bound and solution diversity element libraries are pannedwith fluorescently labeled target protein. The protein targets will bindsome combinations tighter than others, thus directing the evolution ofcombinations to the preferred pairs. DNA barcodes (colored lines), asshown in step 3, may be amplified using universal primers (black lines)to identify individual ligands, which serve as lead molecules.

FIG. 21 is a second embodiment for target screening in accordance withthe present invention. As shown in step 1, two libraries of ligands aresynthesized, one on beads, which may be individually identified throughbarcodes, and the second covalently linked to a DNA barcode (allowingfor stepwise synthesis and identification of the binding ligand). Eachmonomer element consists of a low MW (approx. 600-800) binding ligand(diversity element) covalently linked to a low MW (approx. under 300)linker element (dynamic combinatorial chemistry element). Underphysiological conditions, different combinations of ligands are formingand reassociating with each other through the linker elements. The twolibraries are panned with fluorescently labeled target protein. Theprotein targets will bind some combinations tighter than others, thusdirecting the evolution of combinations to the preferred pairs. In step2, DNA barcodes (colored lines) may be amplified using universal primers(black lines) to identify individual ligands. The top set of theseligands are resynthesized, as shown in step 3, in both bead and solutionformat, with the option of adding additional diversity in the connectorbetween the linker element and the ligand diversity element. The surfacebound and solution diversity element libraries are panned withfluorescently labeled target protein. The protein targets will bind somecombinations tighter than others, thus directing the evolution ofcombinations to the preferred pairs. For step 4, DNA barcodes (coloredlines) may be amplified using universal primers (black lines) toidentify individual ligands, which serve as lead molecules.

FIG. 22 is a third embodiment for target screening in accordance withthe present invention. In step 1, a library of ligands is synthesized,where each monomer element comprises a low MW (approx. 600-800) bindingligand (diversity element) covalently linked to DNA barcode (allowingfor stepwise synthesis and identification of the binding ligand) as wellas a low MW (approx. under 300) linker element (dynamic combinatorialchemistry element). The ligands are allowed to bind to target proteincovalently linked to a solid support. For step 2, DNA barcodes (coloredlines) may be amplified using universal primers (black lines) toidentify individual ligands. The top set of these ligands areresynthesized with the option of adding additional diversity in theconnector between the linker element and the ligand diversity element,as shown in step 3. A second library of ligands is synthesized, whereeach monomer element consists of a low MW binding ligand (diversityelement) covalently linked to a DNA barcode as well as a low MW linkerelement, suitable for reversible binding to the linker element elementin the first library. Under physiological conditions, differentcombinations of ligands are forming and reassociating with each other.The two diversity element libraries are panned with target protein onbeads. The protein targets will bind some combinations tighter thanothers, thus directing the evolution of combinations to the preferredpairs. This enrichment-amplification-resynthesis selection process maybe repeated to identify higher affinity ligand pairs. For step 4, DNAbarcodes (colored lines) may be amplified using universal primers (blacklines) to identify individual ligands, which serve as lead molecules.

FIG. 23 is a fourth embodiment for target screening in accordance withthe present invention. A library of known ligands to a protein targetsuch as a tyrosine kinase is synthesized, as shown in step 1. Theseligands are covalently linked through a diverse number of connectors toDNA barcode (allowing for identification of the binding ligand) as wellas a low MW (approx, under 300) linker element (dynamic combinatorialchemistry element). The ligands are all capable of binding to targetprotein covalently linked to a solid support. In step 2, a secondlibrary of ligands is synthesized, where each monomer element consistsof a low MW binding ligand (diversity element) covalently linked to aDNA barcode as well as a low MW linker element, suitable for reversiblebinding to the linker element in the first library. Under physiologicalconditions, different combinations of ligands are forming andreassociating with each other. The two diversity element libraries arepanned with target protein on beads. The protein targets will bind somecombinations tighter than others, thus directing the evolution ofcombinations to the preferred pairs. (Thisenrichment-amplification-resynthesis selection process may be repeatedto identify higher affinity ligand pairs.) In step 3, DNA barcodes(colored lines) may be amplified using universal primers (black lines)to identify individual ligands, which serve as lead molecules.

FIG. 24 is a fifth embodiment for target screening in accordance withthe present invention. Two libraries of ligands are synthesized in step1, where each monomer element consists of a low MW (approx. 600-800)binding ligand (diversity element) covalently linked to DNA barcode(allowing for stepwise synthesis and identification of the bindingligand) as well as a low MW (approx. under 300) linker element (dynamiccombinatorial chemistry element). The linker element from the firstlibrary binds reversibly with the linker element from the secondlibrary, such that different combinations of ligands are forming andreassociating with each other. The two diversity element libraries arepanned with target protein on beads. The protein targets will bind somecombinations tighter than others, thus directing the evolution ofcombinations to the preferred pairs. In step 2, DNA barcodes (coloredlines) may be amplified using universal primers (black lines) toidentify individual ligands. The top set of these ligands areresynthesized in step 3 with the option of adding additional diversityin the connector between the linker element and the ligand diversityelement. The extra diversity may be encoded in the DNA barcode in aregion not used for generating ligand diversity in the first round ofsynthesis. The two refined diversity element libraries are again pannedwith target protein on beads. The protein targets will bind somecombinations tighter than others, thus directing the evolution ofcombinations to the preferred pairs. Thisenrichment-amplification-resynthesis-selection process is analogous toDarwinian selection on diploid organisms and may be repeated to identifyhigher affinity ligand pairs. DNA barcodes (colored lines) may beamplified using universal primers (black lines) to identify individualligands, which serve as lead molecules.

FIG. 25 is a schematic representation of a system for cycling pH forselection of coferons using cyclic combinatorial chemistry. A Nafion-117membrane separates an upper compartment A from a lower compartment B.Compartment A contains beads, coferons, buffer (such as PIPS, TEEN, orPIPPS), and target protein. The buffer is chosen to provide the desiredpH range based on pKa values for the buffer. Cation and water exchangebetween compartments A and B is mediated by piston pumps A and B.Cations cycle between H⁺ and Na⁺ or other equivalent cation. CompartmentB is used to wash in and out different buffers in reservoirs C-E.Reservoir C contains an aqueous wash solution. Reservoir D contains H⁺or a low pH buffer. Reservoir E contains NaOH (or equivalent base), or ahigh pH buffer. During cycling, ionic strength and amount of bufferremain unchanged in Compartment A.

FIG. 26 is a schematic representation of a system for cycling metal ionsfor selection of metal co-factor coferons using cyclic combinatorialchemistry. A Nafion-117 membrane separates an upper compartment A from alower compartment B. Compartment A contains beads, coferons, buffer(such as PIPS, TEEN, or PIPPS), and target protein. The buffer is chosento provide the desired pH range based on pKa values for the buffer.Cation and water exchange between compartments A and B is mediated bypiston pumps A and B. Cations cycle between Zn²⁺ and Na⁺. Compartment Bis used to wash in and out different buffers in reservoirs C-E.Reservoir C contains an aqueous wash solution. Reservoir D contains H⁺or a low pH buffer. Reservoir E contains NaOH (or equivalent base), or ahigh pH buffer. During cycling, ionic strength and amount of bufferremain unchanged in Compartment A.

FIG. 27 is a schematic representation of a multimeric protein beinginhibited by a coferon monomer that is capable of assembling in to amultimer. Protective antigen (PA) binds to the cellular anthrax receptor(ANTXR). The protective antigen is cleaved by a protease, while a 20 kDafragment (PA₂₀) leaves, a 63 kDa fragment (PA₆₃) remains bound to thereceptor. PA₆₃ self-associates forming a heptamer, [PA₆₃]₇, to which theedema factor (EF) and lethal factor (LF) bind. A coferon monomer thatcan self-assemble (self-recognizing coferon) in to a multimericstructure can bind and inhibit translocation of the EF/LF in to thecell.

FIG. 28 is a schematic representation of a tetrameric protein beingbound by a coferon that can assemble in to tetramers. The coferon dimeris in reversible equilibrium with the monomeric form of the coferon. Themonomer can bind and inhibit the protein monomer by itself. If theprotein monomers assemble to form a tetrameric protein target, thecoferon monomers can bind the individual protein monomers therebyforming a tetrameric coferon.

FIG. 29 is a schematic drawing of a coferon drug with mother-childlinker elements.

FIG. 30 depicts the synthesis of a library of linker elements.

FIG. 31 depicts the synthesis of a library of linker elements.

FIG. 32 shows the synthetic scheme for preparing linker elements boundto or unbound to beads.

FIG. 33 shows the intercalation of a linker element bound to a bead witha linker element not bound to a bead.

FIG. 34 shows the synthesis of a bead bound library of linkerelement-amino acid chlorides.

FIG. 35 shows the synthesis of cyclopentanol amides where the amidegroup is protected.

FIG. 36 shows the synthesis of a library of linker elements with aminoacids forming an ester linkage with the hydroxyl group of cyclopentanolamides.

FIG. 37 shows the synthesis of a coferon monomer having a linker elementcoupled to a diversity element.

FIG. 38 shows 3 distinct areas of structural diversity in the coferonmonomer of FIG. 37.

FIG. 39 shows the synthesis of coferon dimers from coferon monomers.

FIG. 40 is a schematic representation of a system of two coferons (C1,and C2) that bind to orthogonal sites on a protein target (T) and toeach other via non-covalent association between the linker elements.Coferon 1 is illustrated as a green shape, coferon 2 as a blue shape,and the target protein as a white shape. The equilibrium of coferon 1(C1) binding to the target is given by the dissociation constant K_(d1).The equilibrium of coferon 2 (C2) binding to the target is given by thedissociation constant K_(d2). In turn, coferon C2 may bind the complexof C1T (with dissociation constant of K_(d3)), while coferon C1 may bindthe complex of C2T (with dissociation constant of K_(d4)). The C₁-C₂coferon dimer itself is in equilibrium with the target, withdissociation constant of K_(d6). Finally, the coferon dimer willdissociate to form the two monomers, with dissociation constant ofK_(d5).

FIG. 41 is a schematic representation of a system of two coferons (C1,and C2) that bind to orthogonal sites on a protein target (T) and toeach other via covalent association between the linker elements. Coferon1 (C1) is illustrated as a green shape, coferon 2 (C2) as a blue shape,the covalent bond between the two coferons as a red dot, and the targetprotein as a white shape. The covalent association may be eitherreversible, or essentially irreversible. If it is irreversible, theK_(d5) dissociation value will be zero.

FIG. 42 is a schematic representation of a system of two coferons (C1,and C2) that bind to orthogonal sites on a protein target (T) and toeach other. Coferon 1 (C1) is illustrated as a green pentagon linkerelement tethered to a blue hexagon ligand, coferon 2 (C2) as an orangepentagon linker element tethered to a blue hexagon ligand, and thetarget protein as a white shape. The tether represents one or morerotatable bonds between the linker element and the ligand that binds tothe target protein. When a coferon monomer binds to the target, therotatable bonds remain unrestricted. When two coferon monomers bind toeach other, the rotatable bonds also remain unrestricted. Only when thecoferon dimer forms on the target is there a loss of free rotation, andthis has an enthropy cost.

FIG. 43 is a schematic representation of a system of two coferons (C1,and C2) that bind to orthogonal sites on a protein target (T) and bindcovalently to each other. Coferon 1 (C1) is illustrated as a greenpentagon linker element tethered to a blue hexagon ligand, coferon 2(C2) as an orange pentagon linker element tethered to a blue hexagonligand, the covalent bond between the two linker elements as a red dot,and the target protein as a white shape. The covalent association may beeither reversible, or essentially irreversible. If it is irreversible,the K_(d5) dissociation value will be zero.

FIG. 44 is a schematic representation of two coferons (C1, and C2) thatcan enter cells as monomers, and bind to orthogonal sites on an internalprotein target (T) to inhibit its activity. Coferon 1 (C1) isillustrated as a green shape, coferon 2 (C2) as a blue shape, and thetarget protein as a white shape. In this example, the dissociationconstant between the linker elements of the two coferons has been tunedso that approximately half of the coferons are in the dimer state.Although only monomers traverse the cell membrane, once inside, theydimerize and bind to the target protein.

FIG. 45 is a schematic representation of two coferons (C1, and C2) thatcan enter cells as monomers, and bind to orthogonal sites on an internalprotein target (T) to inhibit its activity. Coferon 1 (C1) isillustrated as a green shape, coferon 2 (C2) as a blue shape, thecovalent bond between the two coferons as a red dot, and the targetprotein as a white shape. In this example, the dissociation constantbetween the linker elements of the two coferons has been tuned so thatapproximately half of the coferons are in the dimer state. Although onlymonomers traverse the cell membrane, once inside, they dimerize and bindto the target protein.

FIG. 46 is a schematic representation of two coferons (C1, and C2) thatcan enter cells as monomers, and bind to orthogonal sites on an internalprotein target (T) to inhibit its activity. Coferon 1 (C1) isillustrated as a green shape, coferon 2 (C2) as a blue shape, and thetarget protein as a white shape. In this example, the dissociationconstant between the linker elements of the two coferons has been tunedso that approximately 2% of the coferons are in the dimer state.Although only monomers traverse the cell membrane, once inside, theybind to the target protein, which accelerates formation of the dimer.Since the dimer binds tightly to the protein target, it does notdissociate easily, and the majority of the protein targets are bound.

FIG. 47 is a schematic representation of two coferons (C1, and C2) thatcan enter cells as monomers, and bind to orthogonal sites on an internalprotein target (T) to inhibit its activity. Coferon 1 (C1) isillustrated as a green shape, coferon 2 (C2) as a blue shape, thecovalent bond between the two coferons as a red dot, and the targetprotein as a white shape. In this example, the linker elements of thetwo coferons do not easily come together, so that only a very smallpercentage of the coferons are in the dimer state. Although onlymonomers traverse the cell membrane, once inside, they bind to thetarget protein, which accelerates formation of its own inhibitor. Linkerelement association is via an irreversible covalent bond, such that oncethe dimer forms on the target protein, its dissociation from the targetis very rare.

FIG. 48 is a schematic representation of two coferons (C1, and C2) thatcan enter cells as monomers, and bind to orthogonal sites on an internalprotein target (T) to inhibit its activity. Coferon 1 (C1) isillustrated as a green shape, coferon 2 (C2) as a blue shape, and thetarget protein as a white shape. The total concentrations of C1 and C2are 0.25 μM, the total protein concentration inside the cell is 0.1 μM,about 100,000 targets per cell. The dissociation constants K_(d1) andK_(d2) between coferon C1 and target T, as well as between coferon C2and target T are given at 1 μM. The dissociation constant between thetwo coferons, K_(d5) is given at 10 48 μM.

FIG. 49 is a graph showing the percent target T that is bound by coferondimer C1C2 vs. coferon C1 concentration. The total target [ST] is set at0.1 μM, K_(d1)=K_(d2) is set at 1 μM, K_(d5) is set at 10 μM, and totalcoferon C1 concentration [C1]=total coferon C2 concentration [C2],varying from 0.1 μM up to 5 μM concentration.

FIG. 50 is a schematic representation of two coferons (C1, and C2) thatcan enter cells as monomers, and bind to orthogonal sites on an internalprotein target (T) to inhibit its activity. Coferon 1 (C1) isillustrated as a green shape, coferon 2 (C2) as a blue shape, and thetarget protein as a white shape. The total concentrations of C1 and C2are 2.5 μM, the total protein concentration inside the cell is 1 μM,about 1,000,000 targets per cell. The dissociation constants K_(d1) andK_(d2) between coferon C1 and target T, as well as between coferon C2and target T are given at 1 μM. The dissociation constant between thetwo coferons, K_(d5) is given at 10 μM.

FIG. 51 is a graph showing the percent target T that is bound by coferondimer C1C2 vs. coferon C1 concentration. The total target [ST] is set at1 μM, K_(d1)=K_(d2) is set at 1 μM, K_(d5) is set at 10 μM, and totalcoferon C1 concentration [C1]=total coferon C2 concentration [C2],varying from 1 μM up to 50 μM concentration.

FIG. 52 is a schematic representation of two coferons (C1, and C2) thatcan enter cells as monomers, and bind to orthogonal sites on an internalprotein target (T) to inhibit its activity. Coferon 1 (C1) isillustrated as a green shape, coferon 2 (C2) as a blue shape, and thetarget protein as a white shape. The total concentrations of C1 and C2are 2.5 μM, the total protein concentration inside the cell is 0.1 μM,about 100,000 targets per cell. The dissociation constants K_(d1) andK_(d2) between coferon C1 and target T, as well as between coferon C2and target T are given at 10 μM. The dissociation constant between thetwo coferons, K_(d5) is given at 100 μM.

FIG. 53 is a graph showing the percent target T that is bound by coferondimer C1C2 vs. coferon C1 concentration. The total target [ST] is set at0.1 μM, K_(d1)=K_(d2) is set at 10 μM, K_(d5) is set at 100 μM, andtotal coferon C1 concentration [C1]=total coferon C2 concentration [C2],varying from 0.1 μM up to 5 μM concentration.

FIG. 54 is a schematic representation of two coferons (C1, and C2) thatcan enter cells as monomers, and bind to orthogonal sites on an internalprotein target (T) to inhibit its activity. Coferon 1 (C1) isillustrated as a green shape, coferon 2 (C2) as a blue shape, and thetarget protein as a white shape. The total concentrations of C1 and C2are 2.5 μM, the total protein concentration inside the cell is 1 μM,about 1,000,000 targets per cell. The dissociation constants K_(d1) andK_(d2) between coferon C1 and target T, as well as between coferon C2and target T are given at 10 μM. The dissociation constant between thetwo coferons, K_(d5) is given at 100 μM.

FIG. 55 is a graph showing the percent target T that is bound by coferondimer C1C2 vs. coferon C1 concentration. The total target [ST] is set at1 μM, K_(d1)=K_(d2) is set at 10 μM, K_(d5) is set at 100 μM, and totalcoferon C1 concentration [C1]=total coferon C2 concentration [C2],varying from 1 μM up to 10 μM concentration.

FIG. 56 is a schematic representation of two coferons (C1, and C2) thatcan enter cells as monomers, and bind to orthogonal sites on an internalprotein target (T) to inhibit its activity. Coferon 1 (C1) isillustrated as a green shape, coferon 2 (C2) as a blue shape, and thetarget protein as a white shape. The total concentrations of C1 and C2are 20 μM, the total protein concentration inside the cell is 10 μM,about 10,000,000 targets per cell. The dissociation constants K_(d1) andK_(d2) between coferon C1 and target T, as well as between coferon C2and target T are given at 10 μM. The dissociation constant between thetwo coferons, K_(d5) is given at 100 μM.

FIG. 57 is a graph showing the percent target T that is bound by coferondimer C1C2 vs. coferon C1 concentration. The total target [ST] is set at10 μM, K_(d1)=K_(d2) is set at 10 μM, K_(d5) is set at 100 μM, and totalcoferon C1 concentration [C1]=total coferon C2 concentration [C2],varying from 10 μM up to 50 μM concentration.

FIG. 58 is a schematic representation of two coferons (C1, and C2) thatcan enter cells as monomers, and bind to orthogonal sites on an internalprotein target (T) to inhibit its activity. Coferon 1 (C1) isillustrated as a green shape, coferon 2 (C2) as a blue shape, and thetarget protein as a white shape. The total concentrations of C1 and C2are 25 μM, the total protein concentration inside the cell is 1 μM,about 1,000,000 targets per cell. The dissociation constants K_(d1) andK_(d2) between coferon C1 and target T, as well as between coferon C2and target T are given at 100 μM. The dissociation constant between thetwo coferons, K_(d5) is given at 1,000 μM.

FIG. 59 is a graph showing the percent target T that is bound by coferondimer C1C2 vs. coferon C1 concentration. The total target [ST] is set at1 μM, K_(d1)=K_(d2) is set at 100 μM, K_(d5) is set at 1,000 μM, andtotal coferon C1 concentration [C1]=total coferon C2 concentration [C2],varying from 1 μM up to 50 μM concentration.

FIG. 60 is a schematic representation of two coferons (C1, and C2) thatcan enter cells as monomers, and bind to orthogonal sites on an internalprotein target (T) to inhibit its activity. Coferon 1 (C1) isillustrated as a green shape, coferon 2 (C2) as a blue shape, and thetarget protein as a white shape. The total concentrations of C1 and C2are 50 μM, the total protein concentration inside the cell is 10 μM,about 10,000,000 targets per cell. The dissociation constants K_(d1) andK_(d2) between coferon C1 and target T, as well as between coferon C2and target T are given at 100 μM. The dissociation constant between thetwo coferons, K_(d5) is given at 1,000 μM.

FIG. 61 is a graph showing the percent target T that is bound by coferondimer C1C2 vs. coferon C1 concentration. The total target [ST] is set at10 μM, K_(d1)-=K_(d2) is set at 100 μM, K_(d5) is set at 1,000 μM, andtotal coferon C1 concentration [C1]=total coferon C2 concentration [C2],varying from 10 μM up to 50 μM concentration.

FIG. 62 is a schematic representation of two coferons (C1, and C2) thatcan enter cells as monomers, and bind to orthogonal sites on an internalprotein target (T) to inhibit its activity. The two coferons have a10-fold difference in binding affinity. Coferon 1 (C1) is illustrated asa green shape, coferon 2 (C2) as a blue shape, and the target protein asa white shape. The total concentrations of C1 is at 50 μM, C2 is at 10μM, the total protein concentration inside the cell is 1 μM, about1,000,000 targets per cell. The dissociation constants K_(d1) betweencoferon C1 and target T is given at 100 μM; that between coferon C2 andtarget T is given at 10 μM. The dissociation constant between the twocoferons, K_(d5) is given at 1,000 μM.

FIG. 63 is a graph showing the percent target T that is bound by coferondimer C1C2 vs. coferon C2 concentration. The total target [ST] is set at1 μM, K_(d1) is set at 100 μM, K_(d2) is set at 10 μM, K_(d5) is set at1,000 μM, and total coferon C1 concentration [C1]=50 μM, and totalcoferon C2 concentration [C2], varying from 1 μM up to 50 μMconcentration.

FIG. 64 is a schematic representation of two coferons (C1, and C2) thatcan enter cells as monomers, and bind to orthogonal sites on an internalprotein target (T) to inhibit its activity. The two coferons have a10-fold difference in binding affinity. Coferon 1 (C1) is illustrated asa green shape, coferon 2 (C2) as a blue shape, and the target protein asa white shape. The total concentrations of C1 is at 50 μM, C2 is at 20μM, the total protein concentration inside the cell is 10 μM, about10,000,000 targets per cell. The dissociation constants K_(d1) betweencoferon C1 and target T is given at 100 μM; that between coferon C2 andtarget T is given at 10 μM. The dissociation constant between the twocoferons, K_(d5) is given at 1,000 μM.

FIG. 65 is a graph showing the percent target T that is bound by coferondimer C1C2 vs. coferon C2 concentration. The total target [ST] is set at10 μM, K_(d1) is set at 100 μM, K_(d2) is set at 10 μM, K_(d5) is set at1,000 μM, and total coferon C1 concentration [C1]=50 μM, and totalcoferon C2 concentration [C2], varying from 1 μM up to 50 μMconcentration.

FIG. 66 is a schematic representation of two coferons (C1, and C2) thatcan enter cells as monomers, and bind to orthogonal sites on an internalprotein target (T) to inhibit its activity. Coferon 1 (C1) isillustrated as a green shape, coferon 2 (C2) as a blue shape, and thetarget protein as a white shape. The total concentrations of C1 and C2are 25 μM, the total protein concentration inside the cell is 1 μM,about 1,000,000 targets per cell. The dissociation constants K_(d1) andK_(d2) between coferon C1 and target T, as well as between coferon C2and target T are given at 100 μM. The dissociation constant between thetwo coferons, K_(d5) is given at 100 μM.

FIG. 67 is a graph showing the percent target T that is bound by coferondimer C1C2 vs. coferon C1-Target dissociation constant (K_(d1)). Thetotal target [ST] is set at 1 μM, K_(d5) is set at 100 μM, and totalcoferon C1 concentration [C1]=total coferon C2 concentration [C2], setat 25 μm, and K_(d1)=K_(d2) varying from 1 μM up to 100 μM.

FIG. 68 is a schematic representation of two coferons (C1, and C2) thatcan enter cells as monomers, and bind to orthogonal sites on an internalprotein target (T) to inhibit its activity. Coferon 1 (C1) isillustrated as a green shape, coferon 2 (C2) as a blue shape, and thetarget protein as a white shape. The total concentrations of C1 and C2are 25 μM, the total protein concentration inside the cell is 10 μM,about 10,000,000 targets per cell. The dissociation constants K_(d1) andK_(d2) between coferon C1 and target T, as well as between coferon C2and target T are given at 100 μM. The dissociation constant between thetwo coferons, K_(d5) is given at 100 μM.

FIG. 69 is a graph showing the percent target T that is bound by coferondimer C1C2 vs. coferon C1-Target dissociation constant (K_(d1)). Thetotal target [ST] is set at 10 μM, K_(d5) is set at 100 μM, and totalcoferon C1 concentration [C1]=total coferon C2 concentration [C2], setat 25 μM, and K_(d1)=K_(d2) varying from 1 μM up to 100 μM.

FIG. 70 is a schematic representation of two coferons (C1, and C2) thatcan enter cells as monomers, and bind to orthogonal sites on an internalprotein target (T) to inhibit its activity. Coferon 1 (C1) isillustrated as a green shape, coferon 2 (C2) as a blue shape, and thetarget protein as a white shape. The total concentrations of C1 and C2are 25 μM, the total protein concentration inside the cell is 1 μM,about 1,000,000 targets per cell. The dissociation constants K_(d1) andK_(d2) between coferon C1 and target T, as well as between coferon C2and target T are given at 25 μM. The dissociation constant between thetwo coferons, K_(d5) is given at 1,000 μM.

FIG. 71 is a graph showing the percent target T that is bound by coferondimer C1C2 vs. coferon C1-Target dissociation constant (K_(d1)). Thetotal target [ST] is set at 1 μM, K_(d5) is set at 1,000 μM, and totalcoferon C1 concentration [C1]=total coferon C2 concentration [C2], setat 25 μm, and K_(d1)=K_(d2) varying from 1 μM up to 100 μM.

FIG. 72 is a schematic representation of two coferons (C1, and C2) thatcan enter cells as monomers, and bind to orthogonal sites on an internalprotein target (T) to inhibit its activity. Coferon 1 (C1) isillustrated as a green shape, coferon 2 (C2) as a blue shape, and thetarget protein as a white shape. The total concentrations of C1 and C2are 25 μM, the total protein concentration inside the cell is 10 μM,about 10,000,000 targets per cell. The dissociation constants K_(d1) andK_(d2) between coferon C1 and target T, as well as between coferon C2and target T are given at 25 μM. The dissociation constant between thetwo coferons, K_(d5) is given at 1,000 μM.

FIG. 73 is a graph showing the percent target T that is bound by coferondimer C1C2 vs. coferon C1-Target dissociation constant (K_(d1)). Thetotal target [ST] is set at 10 μM, K_(d5) is set at 1,000 μM, and totalcoferon C1 concentration [C1]=total coferon C2 concentration [C2], setat 25 μM, and K_(d1)=K_(d2) varying from 1 μM up to 100 μM.

FIG. 74 is a schematic representation of two coferons (C1, and C2) thatcan enter cells as monomers, and bind to orthogonal sites on an internalprotein target (T) to inhibit its activity. Coferon 1 (C1) isillustrated as a green shape, coferon 2 (C2) as a blue shape, and thetarget protein as a white shape. The total concentrations of C1 and C2are 25 μM, the total protein concentration inside the cell is 1 μM,about 1,000,000 targets per cell. The dissociation constants K_(d1) andK_(d2) between coferon C1 and target T, as well as between coferon C2and target T are given at 10 μM. The dissociation constant between thetwo coferons, K_(d5) is given at 1,000 μM.

FIG. 75 is a graph showing the percent target T that is bound by coferondimer C1C2 vs. coferon C1-C1 dissociation constant (K_(d5)). The totaltarget [ST] is set at 1 μM, K_(d1)=K_(d2) is set at 10 μM, and totalcoferon C1 concentration [C1]=total coferon C2 concentration [C2], setat 25 μm, and K_(d5) varying from 10 μM up to 1,000 μM.

FIG. 76 is a schematic representation of two coferons (C1, and C2) thatcan enter cells as monomers, and bind to orthogonal sites on an internalprotein target (T) to inhibit its activity. Coferon 1 is illustrated asa green shape, coferon 2 as a blue shape, and the target protein as awhite shape. The total concentrations of C1 and C2 are 25 μM, the totalprotein concentration inside the cell is 10 μM, about 10,000,000 targetsper cell. The dissociation constants K_(d1) and K_(d2) between coferonC1 and target T, as well as between coferon C2 and target T are given at10 μM. The dissociation constant between the two coferons, K_(d5) isgiven at 1,000 μM.

FIG. 77 is a graph showing the percent target T that is bound by coferondimer C1C2 vs. coferon C1-C1 dissociation constant (K_(d5)). The totaltarget [ST] is set at 10 μM, K_(d1)=K_(d2) is set at 10 μM, and totalcoferon C1 concentration [C1]=total coferon C2 concentration [C2], setat 25 μm, and K_(d5) varying from 10 μM up to 1,000 μM.

FIG. 78 is a schematic representation of two coferons (C1, and C2) thatcan enter cells as monomers, and bind to orthogonal sites on an internalprotein target (T) to inhibit its activity. Coferon 1 (C1) isillustrated as a green shape, coferon 2 (C2) as a blue shape, and thetarget protein as a white shape. The total concentrations of C1 and C2are 25 μM, the total protein concentration inside the cell is 1 μM,about 1,000,000 targets per cell. The dissociation constants K_(d1) andK_(d2) between coferon C1 and target T, as well as between coferon C2and target T are given at 100 μM. The dissociation constant between thetwo coferons, K_(d5) is given at 250 μM.

FIG. 79 is a graph showing the percent target T that is bound by coferondimer C1-C2 vs. coferon C1-C2 dissociation constant (K_(d5)). The totaltarget [ST] is set at 1 μM, K_(d1)=K_(d2) is set at 10 μM, and totalcoferon C1 concentration [C1]=total coferon C2 concentration [C2], setat 25 μM, and K_(d5) varying from 10 μM up to 1,000 μM.

FIG. 80 is a schematic representation of two coferons (C1, and C2) thatcan enter cells as monomers, and bind to orthogonal sites on an internalprotein target (T) to inhibit its activity. Coferon 1 (C1) isillustrated as a green shape, coferon 2 (C2) as a blue shape, and thetarget protein as a white shape. The total concentrations of C1 and C2are 25 μM, the total protein concentration inside the cell is 10 μM,about 10,000,000 targets per cell. The dissociation constants K_(d1) andK_(d2) between coferon C1 and target T, as well as between coferon C2and target T are given at 100 μM. The dissociation constant between thetwo coferons, K_(d5) is given at 250 μM.

FIG. 81 is a graph showing the percent target T that is bound by coferondimer C1C2 vs. coferon C1-C2 dissociation constant (K_(d5)). The totaltarget [ST] is set at 10 μM, K_(d1)=K_(d2) is set at 10 μM, and totalcoferon C1 concentration [C1]=total coferon C2 concentration [C2], setat 25 μm, and K_(d5) varying from 10 μM up to 1,000 μM.

FIG. 82 is a schematic representation of two coferons (C1, and C2) thatcan enter cells as monomers, and bind to orthogonal sites on an internalprotein target (T) to inhibit its activity. Coferon 1 (C1) isillustrated as a green shape, coferon 2 (C2) as a blue shape, and thetarget protein as a white shape. To more accurately simulate conditionsin vivo, the steady-state or “renewable” concentrations of C1 and C2 are0.1 μM, reflecting the ability to provide a steady dose of drug overtime. The total protein concentration inside the cell is 0.1 μM, about100,000 targets per cell. The dissociation constants K_(d1) and K_(d2)between coferon C1 and target T, as well as between coferon C2 andtarget T are given at 1 μM. The dissociation constant between the twocoferons, K_(d5) is given at 10 μM.

FIG. 83 is a graph showing the percent target T that is bound by coferondimer C1C2 vs. “renewable” coferon C1 concentration. The total target[ST] is set at 0.1 μM, K_(d1)=K_(d2) is set at 1 μM, K_(d5) is set at 10μM, and steady-state or renewable coferon C1 concentration[C1]=steady-state or renewable coferon C2 concentration [C2], varyingfrom 0.1 μM up to 5 μM concentration.

FIG. 84 is a schematic representation of two coferons (C1, and C2) thatcan enter cells as monomers, and bind to orthogonal sites on an internalprotein target (T) to inhibit its activity. Coferon 1 (C1) isillustrated as a green shape, coferon 2 (C2) as a blue shape, and thetarget protein as a white shape. To more accurately simulate conditionsin vivo, the steady-state or “renewable” concentrations of C1 and C2 are0.1 μM, reflecting the ability to provide a steady dose of drug overtime. The total protein concentration inside the cell is 0.25 μM, about250,000 targets per cell. The dissociation constants K_(d1) and K_(d2)between coferon C1 and target T, as well as between coferon C2 andtarget T are given at 1 μM. The dissociation constant between the twocoferons, K_(d5) is given at 10 μM.

FIG. 85 is a graph showing the percent target T that is bound by coferondimer C1C2 vs. “renewable” coferon C1 concentration. The total target[ST] is set at 0.25 μM, K_(d1)=K_(d2) is set at 1 μM, K_(d5) is set at10 μM, and steady-state or renewable coferon C1 concentration[C1]=steady-state or renewable coferon C2 concentration [C2], varyingfrom 0.1 μM up to 5 μM concentration.

FIG. 86 is a schematic representation of two coferons (C1, and C2) thatcan enter cells as monomers, and bind to orthogonal sites on an internalprotein target (T) to inhibit its activity. Coferon 1 (C1) isillustrated as a green shape, coferon 2 (C2) as a blue shape, and thetarget protein as a white shape. To more accurately simulate conditionsin vivo, the steady-state or “renewable” concentrations of C1 and C2 are0.25 μM, reflecting the ability to provide a steady dose of drug overtime. The total protein concentration inside the cell is 1 μM, about1,000,000 targets per cell. The dissociation constants K_(d1) and K_(d2)between coferon C1 and target T, as well as between coferon C2 andtarget T are given at 1 μM. The dissociation constant between the twocoferons, K_(d5) is given at 10 μM.

FIG. 87 is a graph showing the percent target T that is bound by coferondimer C1C2 vs. “renewable” coferon C1 concentration. The total target[ST] is set at 1 μM, K_(d1)=K_(d2) is set at 1 μM, K_(d5) is set at 10μM, and steady-state or renewable coferon C1 concentration[C1]=steady-state or renewable coferon C2 concentration [C2], varyingfrom 0.1 μM up to 5 μM concentration.

FIG. 88 is a schematic representation of two coferons (C1, and C2) thatcan enter cells as monomers, and bind to orthogonal sites on an internalprotein target (T) to inhibit its activity. Coferon 1 (C1) isillustrated as a green shape, coferon 2 (C2) as a blue shape, and thetarget protein as a white shape. To more accurately simulate conditionsin vivo, the steady-state or “renewable” concentrations of C1 and C2 are0.25 μM, reflecting the ability to provide a steady dose of drug overtime. The total protein concentration inside the cell is 2.5 μM, about2,500,000 targets per cell. The dissociation constants K_(d1) and K_(d2)between coferon C1 and target T, as well as between coferon C2 andtarget T are given at 1 μM. The dissociation constant between the twocoferons, K_(d5) is given at 10 μM.

FIG. 89 is a graph showing the percent target T that is bound by coferondimer C1C2 vs. “renewable” coferon C1 concentration. The total target[ST] is set at 2.5 μM, K_(d1)=K_(d2) is set at 1 μM, K_(d5) is set at 10μM, and steady-state or renewable coferon C1 concentration[C1]=steady-state or renewable coferon C2 concentration [C2], varyingfrom 0.1 μM up to 5 μM concentration.

FIG. 90 is a schematic representation of two coferons (C1, and C2) thatcan enter cells as monomers, and bind to orthogonal sites on an internalprotein target (T) to inhibit its activity. Coferon 1 (C1) isillustrated as a green shape, coferon 2 (C2) as a blue shape, and thetarget protein as a white shape. To more accurately simulate conditionsin vivo, the steady-state or “renewable” concentrations of C1 and C2 are0.25 μM, reflecting the ability to provide a steady dose of drug overtime. The total protein concentration inside the cell is 10 μM, about10,000,000 targets per cell. The dissociation constants K_(d1) andK_(d2) between coferon C1 and target T, as well as between coferon C2and target T are given at 1 μM. The dissociation constant between thetwo coferons, K_(d5) is given at 10 μM.

FIG. 91 is a graph showing the percent target T that is bound by coferondimer C1C2 vs. “renewable” coferon C1 concentration. The total target[ST] is set at 10 μM, K_(d1)=K_(d2) is set at 1 μM, K_(d5) is set at 10μM, and steady-state or renewable coferon C1 concentration[C1]=steady-state or renewable coferon C2 concentration [C2], varyingfrom 0.1 μM up to 5 μM concentration.

FIG. 92 is a schematic representation of two coferons (C1, and C2) thatcan enter cells as monomers, and bind to orthogonal sites on an internalprotein target (T) to inhibit its activity. Coferon 1 (C1) isillustrated as a green shape, coferon 2 (C2) as a blue shape, and thetarget protein as a white shape. To more accurately simulate conditionsin vivo, the steady-state or “renewable” concentrations of C1 and C2 are0.25 μM, reflecting the ability to provide a steady dose of drug overtime. The total protein concentration inside the cell is 0.1 μM, about100,000 targets per cell. The dissociation constants K_(d1) and K_(d2)between coferon C1 and target T, as well as between coferon C2 andtarget T are given at 10 μM. The dissociation constant between the twocoferons, K_(d5) is given at 10 μM.

FIG. 93 is a graph showing the percent target T that is bound by coferondimer C1C2 vs. “renewable” coferon C1 concentration. The total target[ST] is set at 0.1 μM, K_(d1)=K_(d2) is set at 10 μM, K_(d5) is set at10 μM, and steady-state or renewable coferon C1 concentration[C1]=steady-state or renewable coferon C2 concentration [C2], varyingfrom 0.1 μM up to 5 μM concentration.

FIG. 94 is a schematic representation of two coferons (C1, and C2) thatcan enter cells as monomers, and bind to orthogonal sites on an internalprotein target (T) to inhibit its activity. Coferon 1 (C1) isillustrated as a green shape, coferon 2 (C2) as a blue shape, and thetarget protein as a white shape. To more accurately simulate conditionsin vivo, the steady-state or “renewable” concentrations of C1 and C2 are0.25 μM, reflecting the ability to provide a steady dose of drug overtime. The total protein concentration inside the cell is 0.25 μM, about250,000 targets per cell. The dissociation constants K_(d1) and K_(d2)between coferon C1 and target T, as well as between coferon C2 andtarget T are given at 10 μM. The dissociation constant between the twocoferons, K_(d5) is given at 10 μM.

FIG. 95 is a graph showing the percent target T that is bound by coferondimer C1C2 vs. “renewable” coferon C1 concentration. The total target[ST] is set at 0.25 μM, K_(d1)=K_(d2) is set at 10 μM, K_(d5) is set at10 μM, and steady-state or renewable coferon C1 concentration[C1]=steady-state or renewable coferon C2 concentration [C2], varyingfrom 0.1 μM up to 5 μM concentration.

FIG. 96 is a schematic representation of two coferons (C1, and C2) thatcan enter cells as monomers, and bind to orthogonal sites on an internalprotein target (T) to inhibit its activity. Coferon 1 (C1) isillustrated as a green shape, coferon 2 (C2) as a blue shape, and thetarget protein as a white shape. To more accurately simulate conditionsin vivo, the steady-state or “renewable” concentrations of C1 and C2 are0.25 μM, reflecting the ability to provide a steady dose of drug overtime. The total protein concentration inside the cell is 1 μM, about1,000,000 targets per cell. The dissociation constants K_(d1) and K_(d2)between coferon C1 and target T, as well as between coferon C2 andtarget T are given at 10 μM. The dissociation constant between the twocoferons, K_(d5) is given at 10 μM.

FIG. 97 is a graph showing the percent target T that is bound by coferondimer C1C2 vs. “renewable” coferon C1 concentration. The total target[ST] is set at 1 μM, K_(d1)=K_(d2) is set at 10 μM, K_(d5) is set at 10μM, and steady-state or renewable coferon C1 concentration[C1]=steady-state or renewable coferon C2 concentration [C2], varyingfrom 0.1 μM up to 5 μM concentration.

FIG. 98 is a schematic representation of two coferons (C1, and C2) thatcan enter cells as monomers, and bind to orthogonal sites on an internalprotein target (T) to inhibit its activity. Coferon 1 (C1) isillustrated as a green shape, coferon 2 (C2) as a blue shape, and thetarget protein as a white shape. To more accurately simulate conditionsin vivo, the steady-state or “renewable” concentrations of C1 and C2 are0.25 μM, reflecting the ability to provide a steady dose of drug overtime. The total protein concentration inside the cell is 2.5 μM, about2,500,000 targets per cell. The dissociation constants K_(d1) and K_(d2)between coferon C1 and target T, as well as between coferon C2 andtarget T are given at 10 μM. The dissociation constant between the twocoferons, K_(d5) is given at 10 μM.

FIG. 99 is a graph showing the percent target T that is bound by coferondimer C1C2 vs. “renewable” coferon C1 concentration. The total target[ST] is set at 2.5 μM, K_(d1)=K_(d2) is set at 10 μM, K_(d5) is set at10 μM, and steady-state or renewable coferon C1 concentration[C1]=steady-state or renewable coferon C2 concentration [C2], varyingfrom 0.1 μM up to 5 μM concentration.

FIG. 100 is a schematic representation of two coferons (C1, and C2) thatcan enter cells as monomers, and bind to orthogonal sites on an internalprotein target (T) to inhibit its activity. Coferon 1 (C1) isillustrated as a green shape, coferon 2 (C2) as a blue shape, and thetarget protein as a white shape. To more accurately simulate conditionsin vivo, the steady-state or “renewable” concentrations of C1 and C2 are0.25 μM, reflecting the ability to provide a steady dose of drug overtime. The total protein concentration inside the cell is 10 μM, about10,000,000 targets per cell. The dissociation constants K_(d1) andK_(d2) between coferon C1 and target T, as well as between coferon C2and target T are given at 10 μM. The dissociation constant between thetwo coferons, K_(d5) is given at 10 μM.

FIG. 101 is a graph showing the percent target T that is bound bycoferon dimer C1C2 vs. “renewable” coferon C1 concentration. The totaltarget [ST] is set at 10 μM, K_(d1)=K_(d2) is set at 10 μM, K_(d5) isset at 10 μM, and steady-state or renewable coferon C1 concentration[C1]=steady-state or renewable coferon C2 concentration [C2], varyingfrom 0.1 μM up to 5 μM concentration.

FIG. 102 is a schematic representation of two coferons (C1, and C2) thatcan enter cells as monomers, and bind to orthogonal sites on an internalprotein target (T) to inhibit its activity. Coferon 1 (C1) isillustrated as a green shape, coferon 2 (C2) as a blue shape, and thetarget protein as a white shape. To more accurately simulate conditionsin vivo, the steady-state or “renewable” concentrations of C1 and C2 are0.1 μM, reflecting the ability to provide a steady dose of drug overtime. The total protein concentration inside the cell is 0.1 μM, about100,000 targets per cell. The dissociation constant K_(d1) betweencoferon C1 and target T, is given at 1 μM, and the dissociation constantK_(d2) between coferon C2 and target T, is given at 10 μM. Thedissociation constant between the two coferons, K_(d5) is given at 10μM.

FIG. 103 is a graph showing the percent target T that is bound bycoferon dimer C1C2 vs. “renewable” coferon C1 concentration. The totaltarget [ST] is set at 0.1 μM, K_(d1) is set at 1 μM, K_(d2) is set at 10μM, K_(d5) is set at 10 μM, and steady-state or renewable coferon C1concentration [C1]=steady-state or renewable coferon C2 concentration[C2], varying from 0.1 μM up to 5 μM concentration.

FIG. 104 is a schematic representation of two coferons (C1, and C2) thatcan enter cells as monomers, and bind to orthogonal sites on an internalprotein target (T) to inhibit its activity. Coferon 1 (C1) isillustrated as a green shape, coferon 2 (C2) as a blue shape, and thetarget protein as a white shape. To more accurately simulate conditionsin vivo, the steady-state or “renewable” concentrations of C1 and C2 are0.1 μM, reflecting the ability to provide a steady dose of drug overtime. The total protein concentration inside the cell is 0.25 μM, about250,000 targets per cell. The dissociation constant K_(d1) betweencoferon C1 and target T, is given at 1 μM, and the dissociation constantK_(d2) between coferon C2 and target T, is given at 10 μM. Thedissociation constant between the two coferons, K_(d5) is given at 10μM.

FIG. 105 is a graph showing the percent target T that is bound bycoferon dimer C1C2 vs. “renewable” coferon C1 concentration. The totaltarget [ST] is set at 0.25 μM, K_(d1) is set at 1 uM, K_(d2) is set at10 μM, K_(d5) is set at 10 μM, and steady-state or renewable coferon C1concentration [C1]=steady-state or renewable coferon C2 concentration[C2], varying from 0.1 μM up to 5 μM concentration.

FIG. 106 is a schematic representation of two coferons (C1, and C2) thatcan enter cells as monomers, and bind to orthogonal sites on an internalprotein target (T) to inhibit its activity. Coferon 1 is illustrated asa green shape, coferon 2 as a blue shape, and the target protein as awhite shape. To more accurately simulate conditions in vivo, thesteady-state or “renewable” concentrations of C1 and C2 are 0.25 μM,reflecting the ability to provide a steady dose of drug over time. Thetotal protein concentration inside the cell is 1 μM, about 1,000,000targets per cell. The dissociation constant K_(d1) between coferon C1and target T, is given at 1 μM, and the dissociation constant K_(d2)between coferon C2 and target T, is given at 10 μM. The dissociationconstant between the two coferons, K_(d5) is given at 10 μM.

FIG. 107 is a graph showing the percent target T that is bound bycoferon dimer C1C2 vs. “renewable” coferon C1 concentration. The totaltarget [ST] is set at 1 μM, K_(d1) is set at 1 μM, K_(d2) is set at 10μM, K_(d5) is set at 10 μM, and steady-state or renewable coferon C1concentration [C1]=steady-state or renewable coferon C2 concentration[C2], varying from 0.1 μM up to 5 μM concentration.

FIG. 108 is a schematic representation of two coferons (C1, and C2) thatcan enter cells as monomers, and bind to orthogonal sites on an internalprotein target (T) to inhibit its activity. Coferon 1 (C1) isillustrated as a green shape, coferon 2 (C2) as a blue shape, and thetarget protein as a white shape. To more accurately simulate conditionsin vivo, the steady-state or “renewable” concentrations of C1 and C2 are0.25 μM, reflecting the ability to provide a steady dose of drug overtime. The total protein concentration inside the cell is 2.5 μM, about2,500,000 targets per cell. The dissociation constant K_(d1) betweencoferon C1 and target T, is given at 1 μM, and the dissociation constantK_(d2) between coferon C2 and target T, is given at 10 μM. Thedissociation constant between the two coferons, K_(d5) is given at 10μM.

FIG. 109 is a graph showing the percent target T that is bound bycoferon dimer C1C2 vs. “renewable” coferon C1 concentration. The totaltarget [ST] is set at 2.5 μM, K_(d1) is set at 1 μM, K_(d2) is set at 10μM, K_(d5) is set at 10 μM, and steady-state or renewable coferon C1concentration [C1]=steady-state or renewable coferon C2 concentration[C2], varying from 0.1 μM up to 5 μM concentration.

FIG. 110 is a schematic representation of two coferons (C1, and C2) thatcan enter cells as monomers, and bind to orthogonal sites on an internalprotein target (T) to inhibit its activity. Coferon 1 (C1) isillustrated as a green shape, coferon 2 (C2) as a blue shape, and thetarget protein as a white shape. To more accurately simulate conditionsin vivo, the steady-state or “renewable” concentrations of C1 and C2 are0.25 μM, reflecting the ability to provide a steady dose of drug overtime. The total protein concentration inside the cell is 10 μM, about10,000,000 targets per cell. The dissociation constant K_(d1) betweencoferon C1 and target T, is given at 1 μM, and the dissociation constantK_(d2) between coferon C2 and target T, is given at 10 μM. Thedissociation constant between the two coferons, K_(d5) is given at 10μM.

FIG. 111 is a graph showing the percent target T that is bound bycoferon dimer C1C2 vs. “renewable” coferon C1 concentration. The totaltarget [ST] is set at 10 μM, K_(d1) is set at 1 μM, K_(d2) is set at 10μM, K_(d5) is set at 10 μM, and steady-state or renewable coferon C1concentration [C1]=steady-state or renewable coferon C2 concentration[C2], varying from 0.1 μM up to 5 μM concentration.

FIG. 112 is a schematic representation of two coferons (C1, and C2) thatcan enter cells as monomers, and bind to orthogonal sites on an internalprotein target (T) to inhibit its activity. Coferon 1 (C1) isillustrated as a green shape, coferon 2 (C2) as a blue shape, and thetarget protein as a white shape. To more accurately simulate conditionsin vivo, the steady-state or “renewable” concentrations of C1 and C2 are0.5 μM, reflecting the ability to provide a steady dose of drug overtime. The total protein concentration inside the cell is 0.1 μM, about100,000 targets per cell. The dissociation constants K_(d1) and K_(d2)between coferon C1 and target T, as well as between coferon C2 andtarget T are given at 1 μM. The dissociation constant between the twocoferons, K_(d5) is given at 100 μM.

FIG. 113 is a graph showing the percent target T that is bound bycoferon dimer C1C2 vs. “renewable” coferon C1 concentration. The totaltarget [ST] is set at 0.1 μM, K_(d1)=K_(d2) is set at 1 μM, K_(d5) isset at 100 μM, and steady-state or renewable coferon C1 concentration[C1]=steady-state or renewable coferon C2 concentration [C2], varyingfrom 0.1 μM up to 5 μM concentration.

FIG. 114 is a schematic representation of two coferons (C1, and C2) thatcan enter cells as monomers, and bind to orthogonal sites on an internalprotein target (T) to inhibit its activity. Coferon 1 (C1) isillustrated as a green shape, coferon 2 (C2) as a blue shape, and thetarget protein as a white shape. To more accurately simulate conditionsin vivo, the steady-state or “renewable” concentrations of C1 and C2 are0.1 μM, reflecting the ability to provide a steady dose of drug overtime. The total protein concentration inside the cell is 0.25 μM, about250,000 targets per cell. The dissociation constants K_(d1) and K_(d2)between coferon C1 and target T, as well as between coferon C2 andtarget T are given at 1 μM The dissociation constant between the twocoferons, K_(d5) is given at 100 μM.

FIG. 115 is a graph showing the percent target T that is bound bycoferon dimer C1C2 vs. “renewable” coferon C1 concentration. The totaltarget [ST] is set at 0.25 μM, K_(d1)=K_(d2) is set at 1 μM, K_(d5) isset at 100 μM, and steady-state or renewable coferon C1 concentration[C1]=steady-state or renewable coferon C2 concentration [C2], varyingfrom 0.1 μM up to 5 μM concentration.

FIG. 116 is a schematic representation of two coferons (C1, and C2) thatcan enter cells as monomers, and bind to orthogonal sites on an internalprotein target (T) to inhibit its activity. Coferon 1 (C1) isillustrated as a green shape, coferon 2 (C2) as a blue shape, and thetarget protein as a white shape. To more accurately simulate conditionsin vivo, the steady-state or “renewable” concentrations of C1 and C2 are0.25 μM, reflecting the ability to provide a steady dose of drug overtime. The total protein concentration inside the cell is 1 μM, about1,000,000 targets per cell. The dissociation constants K_(d1) and K_(d2)between coferon C1 and target T, as well as between coferon C2 andtarget T are given at 1 μM. The dissociation constant between the twocoferons, K_(d5) is given at 100 μM.

FIG. 117 is a graph showing the percent target T that is bound bycoferon dimer C1C2 vs. “renewable” coferon C1 concentration. The totaltarget [ST] is set at 1 μM, K_(d1)=K_(d2) is set at 1 μM, K_(d5) is setat 100 μM, and steady-state or renewable coferon C1 concentration[C1]=steady-state or renewable coferon C2 concentration [C2], varyingfrom 0.1 μM up to 5 μM concentration.

FIG. 118 is a schematic representation of two coferons (C1, and C2) thatcan enter cells as monomers, and bind to orthogonal sites on an internalprotein target (T) to inhibit its activity. Coferon 1 is illustrated asa green shape, coferon 2 as a blue shape, and the target protein as awhite shape. To more accurately simulate conditions in vivo, thesteady-state or “renewable” concentrations of C1 and C2 are 0.25 μM,reflecting the ability to provide a steady dose of drug over time. Thetotal protein concentration inside the cell is 2.5 μM, about 2,500,000targets per cell. The dissociation constants K_(d1) and K_(d2) betweencoferon C1 and target T, as well as between coferon C2 and target T aregiven at 1 μM. The dissociation constant between the two coferons,K_(d5) is given at 100 μM.

FIG. 119 is a graph showing the percent target T that is bound bycoferon dimer C1C2 vs. “renewable” coferon C1 concentration. The totaltarget [ST] is set at 2.5 μM, K_(d1)=K_(d2) is set at 1 μM, K_(d5) isset at 100 μM, and steady-state or renewable coferon C1 concentration[C1]=steady-state or renewable coferon C2 concentration [C2], varyingfrom 0.1 μM up to 5 μM concentration.

FIG. 120 is a schematic representation of two coferons (C1, and C2) thatcan enter cells as monomers, and bind to orthogonal sites on an internalprotein target (T) to inhibit its activity. Coferon 1 (C1) isillustrated as a green shape, coferon 2 (C2) as a blue shape, and thetarget protein as a white shape. To more accurately simulate conditionsin vivo, the steady-state or “renewable” concentrations of C1 and C2 are0.25 μM, reflecting the ability to provide a steady dose of drug overtime. The total protein concentration inside the cell is 10 μM, about10,000,000 targets per cell. The dissociation constants K_(d1) andK_(d2) between coferon C1 and target T, as well as between coferon C2and target T are given at 1 μM. The dissociation constant between thetwo coferons, K_(d5) is given at 100 μM.

FIG. 121 is a graph showing the percent target T that is bound bycoferon dimer C1C2 vs. “renewable” coferon C1 concentration. The totaltarget [ST] is set at 10 μM, K_(d1)=K_(d2) is set at 1 μM, K_(d5) is setat 100 μM, and steady-state or renewable coferon C1 concentration[C1]=steady-state or renewable coferon C2 concentration [C2], varyingfrom 0.1 μM up to 5 μM concentration.

FIG. 122 is a schematic representation of two coferons (C1, and C2) thatcan enter cells as monomers, and bind to orthogonal sites on an internalprotein target (T) to inhibit its activity. Coferon 1 (C1) isillustrated as a green shape, coferon 2 (C2) as a blue shape, and thetarget protein as a white shape. To more accurately simulate conditionsin vivo, the steady-state or “renewable” concentrations of C1 and C2 are1 μM, reflecting the ability to provide a steady dose of drug over time.The total protein concentration inside the cell is 0.1 μM, about 100,000targets per cell. The dissociation constants K_(d1) and K_(d2) betweencoferon C1 and target T, as well as between coferon C2 and target T aregiven at 10 μM. The dissociation constant between the two coferons,K_(d5) is given at 100 μM.

FIG. 123 is a graph showing the percent target T that is bound bycoferon dimer C1C2 vs. “renewable” coferon C1 concentration. The totaltarget [ST] is set at 0.1 μM, K_(d1)=K_(d2) is set at 10 μM, K_(d5) isset at 100 μM, and steady-state or renewable coferon C1 concentration[C1]=steady-state or renewable coferon C2 concentration [C2], varyingfrom 0.1 μM up to 5 μM concentration.

FIG. 124 is a schematic representation of two coferons (C1, and C2) thatcan enter cells as monomers, and bind to orthogonal sites on an internalprotein target (T) to inhibit its activity. Coferon 1 (C1) isillustrated as a green shape, coferon 2 (C2) as a blue shape, and thetarget protein as a white shape. To more accurately simulate conditionsin vivo, the steady-state or “renewable” concentrations of C1 and C2 are1 reflecting the ability to provide a steady dose of drug over time. Thetotal protein concentration inside the cell is 0.25 μM, about 250,000targets per cell. The dissociation constants K_(d1) and K_(d2) betweencoferon C1 and target T, as well as between coferon C2 and target T aregiven at 10 μM. The dissociation constant between the two coferons,K_(d5) is given at 100 μM.

FIG. 125 is a graph showing the percent target T that is bound bycoferon dimer C1C2 vs. “renewable” coferon C1 concentration. The totaltarget [ST] is set at 0.25 K_(d1)=K_(d2) is set at 10 μM, K_(d5) is setat 100 μM, and steady-state or renewable coferon C1 concentration[C1]=steady-state or renewable coferon C2 concentration [C2], varyingfrom 0.1 μM up to 5 μM concentration.

FIG. 126 is a schematic representation of two coferons (C1, and C2) thatcan enter cells as monomers, and bind to orthogonal sites on an internalprotein target (T) to inhibit its activity. Coferon 1 (C1) isillustrated as a green shape, coferon 2 (C2) as a blue shape, and thetarget protein as a white shape. To more accurately simulate conditionsin vivo, the steady-state or “renewable” concentrations of C1 and C2 are2.5 μM, reflecting the ability to provide a steady dose of drug overtime. The total protein concentration inside the cell is 1 μM, about1,000,000 targets per cell. The dissociation constants K_(d1) and K_(d2)between coferon C1 and target T, as well as between coferon C2 andtarget T are given at 10 μM. The dissociation constant between the twocoferons, K_(d5) is given at 100 μM.

FIG. 127 is a graph showing the percent target T that is bound bycoferon dimer C1C2 vs. “renewable” coferon C1 concentration. The totaltarget [ST] is set at 1 μM, K_(d1)=K_(d2) is set at 10 μM, K_(d5) is setat 100 μM, and steady-state or renewable coferon C1 concentration[C1]=steady-state or renewable coferon C2 concentration [C2], varyingfrom 0.1 μM up to 5 μM concentration.

FIG. 128 is a schematic representation of two coferons (C1, and C2) thatcan enter cells as monomers, and bind to orthogonal sites on an internalprotein target (T) to inhibit its activity. Coferon 1 (C1) isillustrated as a green shape, coferon 2 (C2) as a blue shape, and thetarget protein as a white shape. To more accurately simulate conditionsin vivo, the steady-state or “renewable” concentrations of C1 and C2 arereflecting the ability to provide a steady dose of drug over time. Thetotal protein concentration inside the cell is 2.5 μM, about 2,500,000targets per cell. The dissociation constants K_(d1) and K_(d2) betweencoferon C1 and target T, as well as between coferon C2 and target T aregiven at 10 μM. The dissociation constant between the two coferons,K_(d5) is given at 100 μM.

FIG. 129 is a graph showing the percent target T that is bound bycoferon dimer C1C2 vs. “renewable” coferon C1 concentration. The totaltarget [ST] is set at 2.5 μM, K_(d1)=K_(d2) is set at 10 μM, K_(d5) isset at 100 μM, and steady-state or renewable coferon C1 concentration[C1]=steady-state or renewable coferon C2 concentration [C2], varyingfrom 0.1 μM up to 5 μM concentration.

FIG. 130 is a schematic representation of two coferons (C1, and C2) thatcan enter cells as monomers, and bind to orthogonal sites on an internalprotein target (T) to inhibit its activity. Coferon 1 (C1) isillustrated as a green shape, coferon 2 (C2) as a blue shape, and thetarget protein as a white shape. To more accurately simulate conditionsin vivo, the steady-state or “renewable” concentrations of C1 and C2 are1 μM, reflecting the ability to provide a steady dose of drug over time.The total protein concentration inside the cell is 10 μM, about10,000,000 targets per cell. The dissociation constants K_(d1) andK_(d2) between coferon C1 and target T, as well as between coferon C2and target T are given at 10 μM. The dissociation constant between thetwo coferons, K_(d5) is given at 100 μM.

FIG. 131 is a graph showing the percent target T that is bound bycoferon dimer C1C2 vs. “renewable” coferon C1 concentration. The totaltarget [ST] is set at 10 μM, K_(d1)=K_(d2) is set at 10 μM, K_(d5) isset at 100 μM, and steady-state or renewable coferon C1 concentration[C1]=steady-state or renewable coferon C2 concentration [C2], varyingfrom 0.1 μM up to 5 μM concentration.

FIG. 132 is a schematic representation of two coferons (C1, and C2) thatcan enter cells as monomers, and bind to orthogonal sites on an internalprotein target (T) to inhibit its activity. Coferon 1 (C1) isillustrated as a green shape, coferon 2 (C2) as a blue shape, and thetarget protein as a white shape. To more accurately simulate conditionsin vivo, the steady-state or “renewable” concentrations of C1 and C2 are0.5 μM, reflecting the ability to provide a steady dose of drug overtime. The total protein concentration inside the cell is 0.1 μM, about100,000 targets per cell. The dissociation constant K_(d1) betweencoferon C1 and target T, is given at 1 μM, and the dissociation constantK_(d2) between coferon C2 and target T, is given at 10 μM. Thedissociation constant between the two coferons, K_(d5) is given at 100μM.

FIG. 133 is a graph showing the percent target T that is bound bycoferon dimer C1C2 vs. “renewable” coferon C1 concentration. The totaltarget [ST] is set at 0.1 μM, K_(d1) is set at 1 μM, K_(d2) is set at 10μM, K_(d5) is set at 100 μM, and steady-state or renewable coferon C1concentration [C1]=steady-state or renewable coferon C2 concentration[C2], varying from 0.5 μM up to 5 μM concentration.

FIG. 134 is a schematic representation of two coferons (C1, and C2) thatcan enter cells as monomers, and bind to orthogonal sites on an internalprotein target (T) to inhibit its activity. Coferon 1 (C1) isillustrated as a green shape, coferon 2 (C2) as a blue shape, and thetarget protein as a white shape. To more accurately simulate conditionsin vivo, the steady-state or “renewable” concentrations of C1 and C2 are0.1 μM, reflecting the ability to provide a steady dose of drug overtime. The total protein concentration inside the cell is 0.25 μM, about250,000 targets per cell. The dissociation constant K_(d1) betweencoferon C1 and target T, is given at 1 μM, and the dissociation constantK_(d2) between coferon C2 and target T, is given at 10 μM. Thedissociation constant between the two coferons, K_(d5) is given at 100μM.

FIG. 135 is a graph showing the percent target T that is bound bycoferon dimer C1C2 vs. “renewable” coferon C1 concentration. The totaltarget [ST] is set at 0.25 μM, K_(d1) is set at 1 μM, K_(d2) is set at10 μM, K_(d5) is set at 100 μM, and steady-state or renewable coferon C1concentration [C1]=steady-state or renewable coferon C2 concentration[C2], varying from 0.1 μM up to 5 μM concentration.

FIG. 136 is a schematic representation of two coferons (C1, and C2) thatcan enter cells as monomers, and bind to orthogonal sites on an internalprotein target (T) to inhibit its activity. Coferon 1 (C1) isillustrated as a green shape, coferon 2 (C2) as a blue shape, and thetarget protein as a white shape. To more accurately simulate conditionsin vivo, the steady-state or “renewable” concentrations of C1 and C2 are0.25 μM, reflecting the ability to provide a steady dose of drug overtime. The total protein concentration inside the cell is 1 μM, about1,000,000 targets per cell. The dissociation constant K_(d1) betweencoferon C1 and target T, is given at 1 μM, and the dissociation constantK_(d2) between coferon C2 and target T, is given at 10 μM. Thedissociation constant between the two coferons, K_(d5) is given at 100μM.

FIG. 137 is a graph showing the percent target T that is bound bycoferon dimer C1C2 vs. “renewable” coferon C1 concentration. The totaltarget [ST] is set at 1 μM, K_(d1) is set at 1 μM, K_(d2) is set at 10μM, K_(d5) is set at 100 μM, and steady-state or renewable coferon C1concentration [C1]=steady-state or renewable coferon C2 concentration[C2], varying from 0.1 μM up to 5 μM concentration.

FIG. 138 is a schematic representation of two coferons (C1, and C2) thatcan enter cells as monomers, and bind to orthogonal sites on an internalprotein target (T) to inhibit its activity. Coferon 1 (C1) isillustrated as a green shape, coferon 2 (C2) as a blue shape, and thetarget protein as a white shape. To more accurately simulate conditionsin vivo, the steady-state or “renewable” concentrations of C1 and C2 are0.25 μM, reflecting the ability to provide a steady dose of drug overtime. The total protein concentration inside the cell is 2.5 μM, about2,500,000 targets per cell. The dissociation constant K_(d1) betweencoferon C1 and target T, is given at 1 μM, and the dissociation constantK_(d2) between coferon C2 and target T, is given at 10 μM. Thedissociation constant between the two coferons, K_(d5) is given at 100μM.

FIG. 139 is a graph showing the percent target T that is bound bycoferon dimer C1C2 vs. “renewable” coferon C1 concentration. The totaltarget [ST] is set at 2.5 μM, K_(d1) is set at 1 μM, K_(d2) is set at 10μM, K_(d5) is set at 100 μM, and steady-state or renewable coferon C1concentration [C1]=steady-state or renewable coferon C2 concentration[C2], varying from 0.1 μM up to 5 μM concentration.

FIG. 140 is a schematic representation of two coferons (C1, and C2) thatcan enter cells as monomers, and bind to orthogonal sites on an internalprotein target (T) to inhibit its activity. Coferon 1 (C1) isillustrated as a green shape, coferon 2 (C2) as a blue shape, and thetarget protein as a white shape. To more accurately simulate conditionsin vivo, the steady-state or “renewable” concentrations of C1 and C2 are0.25 μM, reflecting the ability to provide a steady dose of drug overtime. The total protein concentration inside the cell is 10 μM, about10,000,000 targets per cell. The dissociation constant K_(d1) betweencoferon C1 and target T, is given at 1 μM, and the dissociation constantK_(d2) between coferon C2 and target T, is given at 10 μM. Thedissociation constant between the two coferons, K_(d5) is given at 100μM.

FIG. 141 is a graph showing the percent target T that is bound bycoferon dimer C1C2 vs. “renewable” coferon C1 concentration. The totaltarget [ST] is set at 10 μM, K_(d1) is set at 1 μM, K_(d2) is set at 10μM, K_(d5) is set at 100 μM, and steady-state or renewable coferon C1concentration [C1]=steady-state or renewable coferon C2 concentration[C2], varying from 0.1 μM up to 5 μM concentration.

FIG. 142 shows the mass to charge ratios (m/z) for selected linkerelement precursors as well as data obtained from mass spectrometry (MS)and mass spectrometry/mass spectrometry (MS/MS) experiments on theability of these linker element precursors to form dimers or stay asmonomers in solution.

FIG. 143 shows the reversible equilibrium formation of a dimer (a phenylboronate) from linker element precursors (phenylboronic acid and acis-1,2-diol). kD values for this equilibrium for a set of cis-1,2-diolsis shown and range from 10 μM to 206 mM.

DETAILED DESCRIPTION OF THE INVENTION

Basic Principles of Coferon Drugs

Coferons are orally active drugs that can enter cells and, once inside,combine with their partner to interfere with or modulate target proteinactivity. A coferon monomer is composed of a diversity element and alinker element.

In general, coferon drugs contain two ligands that bind to the target,and are held together through their respective linker elementinteractions. In order to assure that the coferon drugs bind to a giventarget, the design of coferon usually incorporates selecting from aknown set of diversity elements and/or synthesizing a wide range ofdiversity elements for one or both of the coferon drug dimer.

Once a coferon dimer has been selected for, or screened by variousassays, it is important to be able to identify the structure of thediversity element. This is especially true under conditions of dynamiccombinatorial chemistry, where dozens to hundreds to thousands or evenmore different diversity elements are being interrogated simultaneouslyin the same well or when binding to a target on a solid surface (i.e.affinity column).

The basic coferon design contains the linker element, which isresponsible for interacting with its partner linker element, and thediversity element, which is responsible for binding to the target. Thelinker element and the diversity element may be directly attached toeach other, or linked together by a connector moiety. The linker elementand/or connector portion may assist in positioning the diversity elementin the ideal conformation or orientation for proper binding to thetarget. In addition, these elements in and of themselves may alsointeract with the target. The encryption element, if used, may beattached to the linker element or the connector portion of the molecule.Ideally, it should be linked to the linker element or connector portionin a manner allowing for easy release or cleavage to remove the DNAportion.

Coferon Monomers

As shown in FIG. 1, the coferon monomers may includes a linker element,a ligand or diversity element, a connector, and a DNA bar code. Thelinker element is a dynamic combinatorial chemistry element which mayhave a molecular weight under 500 daltons, preferably 45-450 daltons; itis responsible for interacting with its partner linker element, and thediversity element. The linker element can have a dissociation constantof 100 nM to 100 μM with or without a co-factor. The ligand or diversityelement is provided to bind to a target molecule and has a molecularweight of about 400 to 800 with a dissociation constant of 100 nM to 100μM. The linker element and the diversity element may be directlyattached to each other or linked together by a connector moiety. Anoptional connector binds the linker element and the ligand or diversityelement, assists in synthesis of the coferon monomer, and may assist inpositioning the diversity element in the ideal conformation ororientation for proper binding to the target. An encryption element,usually a DNA bar code, can be attached to the linker element orconnector for easy release or cleavage. The encryption element isincluded to guide synthesis and to identify coferon monomers; it isremoved from final drug products. FIG. 2.1A is a schematic drawing ofcoferon monomers in accordance with the present invention attached toencoded beads via connectors. FIG. 2.1B is a schematic drawing of acoferon monomer in accordance with the present invention with a DNAbarcode attached through a connector. FIG. 2.1C is a schematic drawingof a coferon dimer attached to an encoded bead via a connector to onemonomer, with a DNA barcode attached to the other monomer. FIG. 2.1D isa schematic drawing of a coferon dimer, with DNA barcodes attached toeach monomer via the connectors. FIG. 2.1E is a schematic drawing of acoferon dimer pursuant to the present invention. FIG. 2.1F is aschematic drawing of coferon monomers in accordance with the presentinvention attached to an encoded bead via the linker element. FIG. 2.1Gis a schematic drawing of a coferon monomer in accordance with thepresent invention with a DNA barcode attached the linker element. FIG.2.1H is a schematic drawing of a coferon dimer attached to an encodedbead via the linker element to one monomer, with a DNA barcode attachedto the other monomer. FIG. 2.1I is a schematic drawing of a coferondimer attached to an encoded bead via linker to one monomer. FIG. 2.1Jis a schematic drawing of a coferon dimer, with DNA barcodes attached toeach monomer via the linker elements. FIG. 2.1K is a schematic drawingof a coferon dimer pursuant to the present invention.

One aspect of the present invention is directed to a monomer useful inpreparing therapeutic compounds. The monomer includes a diversityelement, which potentially binds to a target molecule with adissociation constant of less than 300 μM connected to a linker element,directly or indirectly through a connector, to said diversity element.The linker element has a molecular weight less than 500 daltons and iscapable of forming a reversible covalent bond or non-covalentinteraction with a binding partner of said linker element with adissociation constant of less than 300 μM with or without a co-factorunder physiological conditions.

The monomer can additionally include an encoding element, where thediversity element, the linker element, and the encoding element arecoupled together. The encoding element can be an oligonucleotide or alabeled bead.

Linker Elements

Linker Elements Based on Forming Reversible Imine and Iminium Bonds

The concept of the linker element is to coax two small molecules to bindto one another, taking advantage of hydrophobic, polar, ionic, hydrogenbonding, and/or reversible covalent interactions. The challenge is forthat interaction to be sufficiently strong between the two linkerelements, while simultaneously not so strong between a linker elementand a cellular molecule as to effectively bind and remove the linkerelements from solution.

The substituents on the linker elements can be varied to tune theequilibrium of the reversible association of the linker elements inaqueous solution. For reversible covalent bond formation, linkerelements may be derived from carbonyl groups or boronates.

These linker elements have the advantage of well-documented literatureprecedence for use in dynamic combinatorial chemistry selection.

where X and Y may be varied to tune the equilibrium in aqueous solutionand the lines crossed with a dashed line illustrate the one or morebonds formed joining the one or more diversity elements, directly orthrough a connector, to the molecule. Examples of amines for reversibleamine-carbonyl condensations

Examples of Carbonyl Containing Molecules for Reversible Amine-CarbonylCondensations

Example of Amine-Carbonyl Condensation

There is a high concentration of primary amines free in solution(lysine) and in proteins. Thus, when using a coferon containing aprimary amine, it is important for the companion aldehyde or ketonecontaining coferon to find its partner on the surface of the target. Asan added note of caution, the amine-containing linker element may reactwith sugars when in the aldehyde or ketone tautomeric form. However, ifthe primary amine is beta to a thiol group (which may be in theprotected disulfide form outside the cell), then it has the potential toform an irreversible thiazolidine linker in the final coferon dimer.

where X, Y and Z may be varied to tune the equilibrium in aqueoussolution and the lines crossed with a dashed line illustrate the one ormore bonds formed joining the one or more diversity elements, directlyor through a connector, to the molecule.

Linker Elements Derived from a Carbonyl Group

Linker elements derived from carbonyl groups may participate inreversible hemiacetal and hemiketal formation with alcohols.

where X, may be varied to tune the equilibrium in aqueous solution andthe lines crossed with a dashed line illustrate the one or more bondsformed joining the one or more diversity elements, directly or through aconnector, to the molecule. Linker elements derived from carbonyl groupsmay participate in reversible hydrazone formation with other amines.

Schematic Representation of Reversible Hydrazone Formation

-   -   where R₁,R₂=—F, —Cl, —CF₃, —NO₂, —C═O, —COOH, or other electron        withdrawing group.        Example of Amines for Reversible Hydrazone Formation.

Example of Carbonyl Containing Molecules for Reversible HydrazoneFormation.

Linker Elements Based on Forming Reversible Boronate Esters.

These compounds may be ideal for screening purposes, as well as may workin vivo. One potential caveat is that many sugars have diols that mayreact with the boronic acid containing linker element. Boronates canalso complex with amino alcohols and may also compex with amido acids.

where X, R, R′ and R″may be varied to tune the equilibrium in aqueoussolution and the lines crossed with a dashed line illustrate the one ormore bonds formed joining the one or more diversity elements, directlyor through a connector, to the molecule.

Linker Elements Based on Binding to a Metal Co-Factor

Linker elements that are capable of binding to a bioavailable metal,such as zinc or magnesium should work in vivo.

where X and Y may be varied to tune the equilibrium in aqueous solutionand the lines crossed with a dashed line illustrate the one or morebonds formed joining the one or more diversity elements, directly orthrough a connector, to the molecule.

Linker Elements Derived From Other Functional Groups that Undergo otherReversible Reactions

Linker elements may be derived from functional groups that undergoreversible reactions such as forming Diels-Alder adducts. These havebeen shown to work in dynamic combinatorial chemistry screens (Bout, P.J et al., Organic Lett 7:15-18 (2005), which is hereby incorporated byreference in its entirety).

where X═CN or CO₂R and the lines crossed with a dashed line illustratesthe bonds formed joining the one or more diversity elements to themolecule.

The most sophisticated designs encourage heterodimer linker elementformation, such that A-B pairs are preferred over A-A or B-B homodimerformation. Nevertheless, a successful linker element design that bindstightly to an identical linker element with a different ligand may alsobe used. If the ligands do not influence self-binding, then using twodifferent ligands should generate the A-B heterodimer approximately halfof the time.

One class of linker elements involve covalent interactions that occurand are reversible under physiological conditions. These are S—Sdisulfide bonds, alcohol to ketone to form hemi-ketals, and thiol toketone to form hemi-thioketals.

An important variation in the linker element design is to have thelinker element come together through two covalent bonds. The advantageof such an approach is that even though the individual reaction may beunfavored, once a single bond is made, the local concentration of theother two groups favors formation of the second covalent bond and helpsdrive the equilibrium towards linker element formation.

Some linker element designs may allow linker elements to bind to eachother with minimal or no added binding help from the diversity elements.Such designs include linker elements that bind to each other with theaid of a metal cofactor. These designs expand the potential uses ofcoferons.

Some linker elements may be designed to associate irreversibly withinthe cell. For example, the two linker elements may have maltol or2-picolinic acid groups to chelate Zn²⁺ co-factors. In addition, one mayhave a boronic acid group, and the other an alcohol group. When the twolinker elements are brought in close proximity by the presence of thezinc, the boronic ester forms easily. Either the boronic acid or thezinc chelation linkages by themselves are reversible, but once broughttogether the concentration of the other group is sufficiently high tokeep the linker elements together.

A second and related concept is to prevent or minimize side reactionsbetween the individual linker element and active groups on proteins,amino acids, or other molecules in the cell. Such side reactions may bereduced by designing linker element structures that may be stericallyhindered when reacting with a large macromolecule, but more amenable toreacting when aligned with a partner linker element.

A third concept is to bring two linker elements together due tohydrophobic or other non-covalent affinities, and this increasedproximity allows for a single or double covalent linkage to occur.

Further, the architecture of the linker element covalent interactionsshould favor intermolecular bond formation over intramolecular bondformation.

Another embodiment of the linker element is an aromatic compound, where,when the linker element and its diversity element are bound together,one or more aromatic rings of the binding partner are stacked to guideformation of one or more covalent bonds between the linker element andthe binding partner.

Finally, when the linker elements are in use, they will each have anaffinity to their target, and this too will help assemble the dimericlinker element structure. In other words, the intended target helpsassemble its own inhibitor.

There are four categories of linker elements that form dimers. Theseinclude linker elements that bind reversibly with each other in thepresence of target, linker elements that bind essentially irreversiblywith each other once they are brought in proximity by the target, linkerelements that bind reversibly with each other independent of target, andlinker elements that bind essentially irreversibly with each otherindependent of target.

Derivatives Based on 1,3-Dihydroxyacetone

Derivatives based on 1,3-dihydroxyacetone (Linker Element 1) would mostlikely require bulky blocking groups to reduce the natural reactivity ofthe keto group. Nevertheless, this is the minimal linker element design.

One embodiment of the linker element is an aliphatic compound with ahydroxy group alpha, beta, or gamma to a carbonyl group, where thelinker element and its binding partner, when bound together, form a 6 or8 member di-hemiacetal or di-hemiketal rings, the linker element (i.e.Linker Element 1) is

Generic Structure

where

the lines crossed with a dashed line illustrate the one or more bondsformed joining the one or more diversity elements, directly or through aconnector, to the molecule of Formula (I). If there is no diversityelement at that position, the group may be chosen from the following:—H, —OH or —CH₃.

One example of this embodiment is 1,3-dihydroxyacetone (MW: 90) whichnaturally dimerizes under physiologic conditions.

Another linker element design is based on 2-hydroxycyclohexanone (LinkerElement 2). This linker element makes a rigid 3-ringed structure thatmay be ideal. Different adjacent substitutions may activate the hydroxylgroup or the ketal to favor intermolecular interactions. The molecularweight of 2-hydroxycyclohexanone is 114.

Generic Structure

where

R₁═H, —OH, —CH₃, —F, —CF₃, or another electron withdrawing functionalgroup,

the line crossed with a dashed line illustrates the bond formed joiningthe diversity element, directly or through a connector, to the moleculeof Formula (II) and Formula (III).

Examples of this embodiment of the linker elements are as follows:

-   2A)

-   2B)-   See FIGS. 6-7 for three-dimensional depictions of these linker    elements.-   2C)

The imidazole group may function as an internal catalyst, through protondonation to the opposite linker element or by metal ion catalysis tofacilitate reversible dimer formation.

-   2D)

The bicyclo ring provides steric hindrance and directs attack from thebottom side of the ring. The amine will lead to imine formation andcatalyze the condensation reaction. The protonated amino group mayfunction as an internal catalyst to facilitate reversible dimerformation by acting as a potential proton donor to the opposite linkerelement.

-   2E)

Derivatives Based on Cyclopentane Scaffold with a Strained 5-MemberedHemiacetal or Hemiketal Ring

Another embodiment of the linker element is based on a cyclopentanescaffold (Linker Element 3), where one or more sides is also part of astrained 5-membered hemiacetal ring. R₁ (at ring position 1) and R₃ (atring position 2) are aldehyde or keto groups. R₂ (at ring position 1)and R₄ (at ring position 2) are either hydroxyl or alkoxy groups. Thediversity element may be attached to either position 3, 4, or 5 of thecyclopentane ring. The ring will be in equilibrium between the aldehydeand hemiacetal state. When two such linker elements in the aldehydestates are brought in close proximity, they may form 2 or even 4intermolecular bonds. Additional hydroxyl residues may also be used toallow for formation of intermolecular hemiacetals, such as the morefavored 6 membered rings. Linker element dimers with 4 intermolecularbonds may be exceedingly stable.

Generic Structure

where

R₁=R₃=—H,

R₂=R₄=−H, —OH, or —CH₂OH

the line crossed with a dashed line illustrates the bond formed joiningthe diversity element, directly or through a connector, to the moleculeof Formula (IV).

Examples of this embodiment of linker elements are shown below as items3A) to 3C).

-   3A) Intramolecular Hemiacetal to Intermolecular 6-Membered Diacetal    Ring

-   See FIG. 8 for a 3-dimensional depiction.-   3B) Intramolecular Hemiacetals to Two Intermolecular 6-Membered    Diacetal Rings

-   3C) Two Strained 5-Membered Ring Hemiacetals are in Equilibrium with    the Free Aldehyde Form of the Linker Element.

Derivatives Based on 5-Hydroxy-2-oxo-1-hexanal Structure

Another embodiment of the linker element is based on 5-hydroxy,2-oxo-1-hexanal (Linker Element 4). This compound and its family ofderivatives (see drawings below) also create rigid 3-ringed structures,analogous to the 2-hydroxycyclohexanone dimers. There are multipleopportunities to modulate the reactivity of various groups. Reactivitycan be modulated by the addition of hydroxyl groups (3 and or 5positions of the linear form), while methyl (or other bulky)substituents (3 position of the linear form) provide steric hindrance toprotect the reactive centers from attack by extraneous electrophiles andnucleophiles. Substituents, such as carboxamide or imidazole, may beadded to the 6 position (of the linear form) to enhance reactivity ofthe linker element. Diversity elements may be added to the 6 position(of the linear form).

Generic Structure

where

R₁=R₂=—H, —OH, —CH₃, —F, or —CF₃

R₃=—H, —CH₂NH₂,

Examples of this embodiment of linker elements are shown below as items4A) to 4L).

-   4A)

-   4B)

Addition of methyl groups at 3-position direct nucleophilic attack bythe hydroxyl group from one direction only.

-   4C) Replacing one of the methyl groups at the 3-position with a    hydroxyl group makes the carbonyl more reactive.

-   4D) The 3,5-dihydroxy-2-oxohexanal derivative is predicted to be    very active and can form dimers in two different orientations.

-   4E) Addition of an acetamido group facilitates intermolecular dimer    formation. The amide can act as a proton donor to the opposite    linker element when both linker elements are in the correct    orientation, functioning as an internal catalyst.

-   4F) Replacing one of the methyl groups at the 3 position with a    hydroxyl makes the carbonyl more reactive.

-   4G) Addition of an imidazole substituent to function as an internal    catalyst, facilitates reversible formation of the dimer by acting as    a potential proton donor to the opposite linker element. The two    linker elements need to be in the correct orientation for dimer    formation. (Structures 4G through 4L)

-   4H) Dimethyl substitution at the 3-position provides steric    hindrance to protect the carbonyl from extraneous nucleophiles.    Imidazole group at the 5-position may function as an internal    catalyst to facilitate reversible dimer formation by acting as a    potential proton donor to the opposite linker element.

-   4I) Replacing one of the methyl groups at the 3 position with a    hydroxyl makes the carbonyl more reactive.

-   4J) The carbonyl is flanked by two hydroxyl groups to increase its    reactivity

-   4K) Addition of a methyl group to the 3-position adds steric    hindrance

-   4L) Dimethyl substitution at the 4 position provides steric    hindrance.

Derivative Based on Using an Aromatic Heterocyclic or Non-HeterocyclicRing

Derivatives (Linker Element 5) based on using an aromatic heterocyclicor non-heterocyclic ring with at least one bond to a diversity elementdirectly or through a connector, said ring comprising of 5 or 6 memberedrings either singly or fused together including but not limited tobenzene, naphthalene, purine, pyrimidine, or other aromatic structureswith varying degrees of solubility in aqueous vs. lipid bilayer to bringtwo linker elements together, where the linker elements containadditional alcohol, thiol, boronic acid, aldehyde, or ketone groupswhose proximity (once the hydrophobic surfaces align) favors formationof one or more covalent bonds. This architecture allows for selection oflinker elements that would have suboptimal alignment of the aromaticsurfaces if forming self-dimers and, consequently, will be driven toform heterodimers. Further, for some of the structures, it will beadvantageous for the two reactive groups to be attached to the aromaticring pointing away from each other (but not necessarily in the paraorientation) so they cannot form intramolecular bonds. In the examplesbelow, n=1-3 and m=0-2. Six membered aromatic rings may have aliphaticthiol or alkoxy groups at positions 1 or 3 of the ring and aliphaticaldehyde or ketone substituents at position 3. Diversity elements may beadded to positions 5 or 6 of the ring or may be appended to a C-atombelonging to R₁, R₂, or R₃. Additionally, the six-membered ring maycontain N, O, or S atoms. Linker elements based on two aromatic rings,such as naphthalene, may have aliphatic thiol or alkoxy groups atpositions 1, 3, or 8 of the ring and aliphatic aldehyde or ketonesubstituents at positions 1, 3, or 8 of the ring. Diversity elements maybe added to positions 2, 5, or 7 of the ring or may be appended to aC-atom belonging to R₁, R₂, or R₃. Additionally the aromatic rings maycontain N, O, or S atoms.

where

R₁=—H, —(CH₂)_(n)OH, —(CH₂)SH, B(OH)₂

n=0-3,

R₂ and R₃=—(CH₂)_(n)OH, —NH-Aliphatic

Examples of this embodiment of linker elements are shown below as items5A) to 5J).

-   5A). The general format of Linker Element 5A involves a first linker    element of the formula HS—(CH₂)_(n)-Aromatic-(CH₂)_(n)—C═O(OH) and a    second linker element of the formula    HS—(CH₂)_(n)-Aromatic-(CH₂)_(n)—CH₃. The following structures are    specific examples of this embodiment.

-   5B) The general format of Linker Element 5B involves a first linker    element of the formula

and a second linker element of the formula

The following structures are specific for this embodiment.

-   5C) The general format of Linker Element 5C involves a first linker    element of the formula HS—(CH₂)_(m)-Aromatic-(CH₂)_(m)—C═O    (CH₂)_(n)—OH and a second linker element of the formula    HS—(CH₂)_(n)-Aromatic-CH₃. The following structures are specific    examples of this embodiment.

-   5D) HS—(CH₂)_(m)-Aromatic-(CH₂)_(m)—C═O-Aromatic-(CH₂)_(n)—OH and a    second linker element of the formula HS—(CH₂)_(n)—CH₃. The following    structures are specific examples of this embodiment.

-   5E) The general format of Linker Element 5E involves a first linker    element of the formula    HS—(CH₂)_(m)-Aromatic-(CH₂)_(m)—C═O-Aromatic-(CH₂)_(n)—OH and a    second linker element of the formula HS—(CH₂)_(n)—CH₃. The following    structures are specific examples of this embodiment.

-   5F) The general format of Linker Element 5F involves a first linker    element of the formula    HO—(CH₂)_(m)-Aromatic-(CH₂)_(m)—C═O-Aromatic-(CH₂)_(m)—OH and a    second linker element of the formula O═C—(CH₂)_(m)—CH₃. The    following structures are specific examples of this embodiment.

-   5G) The general format of Linker Element 5G involves a first linker    element of the formula    HS—(CH₂)_(m)-Aromatic-(CH₂)_(m)—C═O-Aromatic-(CH₂)_(m)—SH and a    second linker element of the formula O═C—(CH₂)_(m)—CH₃. The    following structures are specific examples of this embodiment.

-   5H) The general format of Linker Element 5H involves a first linker    element of the formula HO—(CH₂)_(n)-Aromatic-B(OH)₂ and a second    linker element of the formula (HO)₂B-Aromatic-(CH₂)_(n)—OH. The    following structures are specific examples of this embodiment.

-   5I) The general format of Linker Element 51 involves a first linker    element of the formula HO—(CH₂)_(n)-Alicyclic-B(OH)₂ and a second    linker element based on 2-hydroxycyclohexanone. The following    structures are specific examples of this embodiment.

-   5J) The general format of Linker Element 5J involves a first linker    element of the formula CH3—(O═C)-Aromatic-B(OH)₂ and a second linker    element based on Aromatic-NH-Aliphatic. The following structures are    specific examples of this embodiment.

The above derivatives (i.e., linker elements) can also be provided withadditional potential to form non-covalent interactions between thecovalent linker elements and/or aromatic rings, such as hydrophobicinteractions, or hydrogen bonding, or polar, or charge interactions.These additional interactions (Linker Element 6) may help guide therings together in the proper orientation, or may help guide formation ofheterodimer linker elements.

The above derivatives (i.e. linker elements) can also be provided withan additional hydroxyl (alcohol) on the first and the second linkerelements to form intramolecular—but sterically strained—hemiacetal. Forexample, an aromatic structure with one side as part of a 5-memberedhemiacetal ring will be strained. These intramolecular structures(Linker Element 7) are in equilibrium between the aldehyde andhemiacetal state. Should the aldehyde from the linker element react witha lysine amino group or tyrosine hydroxyl group from a cellular protein,it may be more easily released from the protein by formation of theintramolecular hemiacetal. When the first linker element is brought inclose proximity with the second linker element which contains ahydroxyl, there is a balance between forming an intermolecularhemiacetal vs. an intramolecular hemiacetal. Each linker element canform an intermolecular hemiacetal, and, thus, the presence of twointermolecular hemiacetals in the dimer is favored over formation ofseparate intramolecular hemiacetals for each monomer.

where

R₁=—(CH₂)_(n)OH, —(CH₂)_(n)SH or B(OH)₂

n=0-3

Examples of this embodiment of linker elements are shown below as items7A) to 7D).

-   7A)

-   7B)

-   7C)

-   7D)

Derivatives Based on Using Base Pairs of 1,3-Diaminopyrimidine,Diaminopyridine or Aminopyrimidine with Cytosine or 5-Amino-2-Pyridone.

These derivatives may include a log p Tuner. A log tuner is anyaliphatic or aromatic group containing substituents that when added tothe linker element changes the overall log p of the molecule. The1,3-diaminopyrimidine, diaminopyridine or aminopyrimidine, cytosine or5-amino-2-pyridone base pair idea is based on enhancing nucleotide-likebase-pairing with hydrophobic surfaces. While these linker elements(Linker Element 8) may not come together in the absence of target, onceformed in the presence of target they may be rather stable. Thenucleobases may be connected using peptide nucleotide analogue, peptideor other backbone allowing for base pairing and base stacking.

-   where Q is an aromatic heterocyclic or non-heterocyclic ring, said    ring comprising of 5 or 6 membered rings either singly or fused    together, including but not limited to benzene, naphthalene, purine,    pyrimidine, triazine.-   where P is an aromatic or aliphatic group used to modulate the    polarity of the molecule,-   where R₁, R₅=—NH₂, —OH, (CH₂)_(n)OH, —B(OH)₂, or

-   where R₂, R₄=—(CH₂)_(n)—, —O(CH₂)_(n)—, —(CH₂)_(n)O—, —NH(CH₂)_(n),    —(CH₂)_(n)NH—, H

-   where R₃=

-   where n=0-3,    where the lines crossed with a dashed line illustrate the bonds    formed joining the one or more diversity elements of the molecule of    Formula (XIII) or (XIV).

Examples of this embodiment of linker elements are shown below as items8A) to 8H).

-   (i) A single base pair, each with an aliphatic log p Tuner that go    above and below the bases.-   8A)

-   8B)

-   8C)

-   8D)

-   A single base pair, each with an aromatic log p Tuner, both go above    the bases, allowing for covalent bonding between an aldehyde/ketone    on one aromatic ring with an alcohol on the other ring.-   8E)

-   8F)

-   8G)

-   8H)

Derivatives Based on Forming Dimers of Nucleobases of,1,3-Diaminopyrimidine, Diaminopyridine or Aminopyrimidine Base Pairswith Cytosine or 5-Amino-2-Pyridone.

These linker elements (Linker Element 9) are based on enhancingnucleotide base-pairing with hydrophobic surfaces. Each bottomnucleotide can form 3 hydrogen bonds with its pair. Each top nucleotidecan form 2 hydrogen bonds with its pair, and is attached through ahemiacetal linker. The top nucleotides can become covalently linkedacross the hydrogen bond area in the “major groove”. While these linkerelements may not come together in the absence of target, once formed inthe presence of target they may be stable.

Examples of this embodiment of linker elements are shown below as items9A) to 9B)

-   9A) 4-aminopyrimidine:5-amino-2-pyridone base pair

-   9B): 5-amino-2-pyridone:6-fluorouracil base pair.

A linear peptide based backbone may be used for coferon monomers withbase-pairing nucleobase linker elements. The diversity elements in thesecoferon monomers may be derived from alternating amino acid residues,while the nucleobases serve as the linker element elements thatassociate with the nucleobase linker element elements of another coferonmonomer.

Linear Form of a Peptide Backbone for Coferon Monomer Elements

In another embodiment a circularized peptide nucleic acid (PNA) basedbackbone for coferon monomers may be used with base-pairing nucleobaselinker elements. The diversity elements in these coferon monomers may bederived from adjacent amino acid residues, while the nucleobases serveas the linker element elements that associate with the nucleobase linkerelement elements of another coferon monomer. A final spacer linking thePNA to the peptide portion may be utilized to relieve ring strain.

In another embodiment, a linear version of a backbone based on using thecyclopentane scaffold may be used for the coferon monomers. The choiceof nucleobase and the substitutions determine whether base stacking orintercalation is preferred.

Derivatives Based on Forming Heterocycle Dimers using Boric Acid Estersto Link the Nucleotides.

In this embodiment, covalent bonds are formed on both the upper andlower base pair. Four hydrogen bonds and two covalent bonds are formedto create a very stable dimer. For this linker element design, duringthe screening process under dynamic combinatorial conditions, only onecovalent bond either on the top or bottom base pair would be allowed.When the final coferons are synthesized in the therapeutic form covalentbonds would be allowed for both the top and bottom base pairs.

Examples of this embodiment of linker elements are shown below as items10A) to 10C).

-   10A)

-   10B)

-   10C)

Derivatives Based on Linker Elements that Bind Each Other with Aid of aCofactor.

These linker elements (Linker Element 11) form mixed dimers throughbidentate binding to zinc or other bioavailable metal ions. Once formed,these linker elements bind to each other very tightly, and can dimerizein the absence of target. Thus, these linker elements may be used tobring together two different drug molecules into cancer cells toincrease their biological potency. For these linker elements, thediversity elements do not need to bind to the same target, although theywill accelerate formation of the dimers if they do have affinity for thesame target. These linker elements will be monomers in regions of thebody with low levels of zinc, but may shift towards the dimer form incells that have higher zinc content. Since cancer cells have increasedexpression of zinc transporter, this provides an additional opportunityfor targeting these drugs to cancer cells.

-   where R═H, B(OH)₂,

-    (CH₂)_(n)OH and n=1-3    -   where R=—CH(OH)—(CH₂)_(m)OH and m=1-2        where the lines crossed with a dashed line illustrate the bonds        formed joining the one or more diversity elements to, directly        or through a connector, the molecule of Formula (Z1).

Examples of this embodiment of linker elements are shown below as items11A) to 11C). The simplest form is based on derivatives of picolinicacid. This version is likely to be highly reversible, and will probablynot dimerize in the absence of target at the concentrations of Zn⁺² andcoferons found inside cells.

-   11A)

-   11B)

The more sophisticated versions of these dimers have either two Zncations, or one Zn cation and a second linking group, such as a boricacid ester

-   11C)

-   11D)

Derivatives Based on Using Two Aromatic Rings Separated by a Rigid Line

Derivatives Based on Using Two Aromatic Rings, such as benzene,naphthalene, purine, pyrimidine, or other aromatic structures, withvarying degrees of solubility in aqueous vs. lipid bilayer to bring twolinker elements together, by favoring overall solubility in aqueoussolution when aromatic surfaces interact (Linker Element 12). Thepreferred version of such a linker element has one aromatic ring that ispoorly soluble in aqueous liquids (i.e. benzene, naphthalene),covalently linked through a partially rigid linker to a second aromaticring that is more soluble in aqueous liquids (i.e., purine, pyrimidine).The partially rigid linker has a geometry that prevents optimal stackingof the two aromatic rings in an intramolecular fashion. However, thepartially rigid linker allows for spacing of the two aromatic rings sothey can intercalate with a partner linker element such that the fouraromatic systems are stacked on top of one another. For example, iflinker element A consisted of pyrimidine-linker-benzene, and linkerelement B consisted of benzene-linker-pyrimidine, then the stackingwould be pyrimidine(A)-benzene(B)-benzene(A)-pyrimidine(B).

Derivatives Based on Using Aromatic Rings that Intercalate with TheseBinding Partners.

An additional embodiment of the linker element (Linker Element 9) is anaromatic compound which intercalates with one or more of its bindingpartner(s), such that intercalation guides formation of zero, one, ormore covalent bonds between the linker element and the binding partner.

Generic Structure

-   where Q is an aromatic heterocyclic or non-heterocyclic ring, said    ring comprising of 5 or 6 membered rings either singly or fused    together.    where the lines crossed with a dashed line illustrate the bonds    formed joining the one or more diversity elements to, directly or    through a connector, the molecule of Formula (XIII)

Examples of this embodiment of linker elements (i.e. Linker Element 13)are as follows:

Derivatives Based on Using an Aromatic Rings that Intercalate with TheirBinding Partners, with Additional Interactions.

Derivatives based on using two aromatic rings can be provided withadditional potential for the two linker elements (i.e. Linker Element14) to form non-covalent interactions between the partially rigid linkerelements and/or aromatic rings, such as hydrophobic interactions, orhydrogen bonding, or polar, or charge interactions. Examples of thisembodiment are set forth as items 14A) to 14B) below.

-   14A)

-   14B)

-   See also FIGS. 9-13.

In a further embodiment (i.e. Linker Element 15), the linker element hasone or more active moieties in a protected state suitable fordeprotection once inside the body, cell, or cellular compartment. Theprotected states include disulfide protection of a reactive thiol group,ester protection of a reactive alcohol group, hemiacetal protection of areactive aldehyde group, hemiketal protection of a reactive ketone groupor alcohol protection of a reactive boronate group.

Derivatives based on using two aromatic rings can be provided withadditional potential for the two linker elements to form one or morereversible covalent bonds between the partially rigid linker elementsand/or aromatic rings, such as S—S disulfide bonds, alcohol to aldehydeor ketone to form hemiacetals or hemiketals. Examples of this embodimentare set forth as Items 15A) to 15B) below.

-   15A)

-   15B)

Derivatives Based on Combined Simple Linker Elements

Any of the preceding linker elements (i.e. Linker Elements 1-14) can beprovided with potential to form complexes of more than two linkerelements. This is Linker Element 16. For example, Linker Element 5,where aromatic rings stack on top of each other, may be designed tostack A-B-C. A may link to'B and C below. B links to A above and Cbelow, and C links to B and A above. Alternatively, Linker Element 5would have two links from A to B, and two additional links from B to C.For Linker Element 7, the partially rigid linker of the central linkerelements would allow stacking of two aromatic rings in between them,while the end linker elements would only permit stacking of one aromaticring between them. For combining 3 such linker elements, the designwould be linker element A consisting of pyrimidine-linker-benzene,linker element B consisting of benzene-linker (that is slightlylonger)-benzene, and linker element C consisting ofbenzene-linker-pyrimidine, then the stacking of the aromatic rings wouldbepyrimidine(A)-benzene(B)-benzene(A)-benzene(C)-benzene(B)-pyrimidine(C).

-   16A)

Monomers Reaction Mechanism

-   16B)

Monomers Reaction Mechanism

Trimers

Linker elements that form multimers are divided in to foursubcategories. These include linker elements that bind reversibly witheach other in the presence of target, linker elements that bindessentially irreversibly with each other once they are brought inproximity by the target, linker elements that bind reversibly with eachother independent of target and linker elements that bind essentiallyirreversibly with each other independent of target.

Linker elements that are part of multimeric structures may be designedin three basic formats as follows: (i) linker elements that dimerize,and then have the ability to form higher order structures; (ii) linkerelements where one linker element has the ability to bind two or morelinker elements, i.e. to make A-B-A trimers; and (iii) linker elementswith the ability to make two or more covalent bonds to other linkerelements, but in a geometry that inhibits two or more of those bondsgoing to the same linker element partner.

Derivatives Based on Hydrogen and/or Covalent Linking of Aromatic“Bases” that form Hexameric Multimers

The derivatives based on hexamers of uracil, diaminotriazine,triaminotriazine, 2,6-diaminopurine, 1,3-diaminopyrimidine,diaminopyridine, and 1,3-diaminopyrimidine bases.

i) Linker elements based on 6 “bases” forming a circular hexagonalplanar structure through hydrogen bonding, each with an aliphatic log pTuner that go above the bases (Linker Element 17).

Where R=an aliphatic group that modulates logP and serves as a logPtuner.

(ii)) Linker elements based on 6 “bases” forming a circular hexagonalplanar structure through hydrogen bonding, each with an aromatic log pTuner that go above the bases, allowing for covalent bonding between analdehyde/ketone on one side of the aromatic ring with an alcohol on theadjacent ring (Linker Element 18). The aldehyde/ketone and alcoholgroups are in the meta orientation on the aromatic ring (120°orientation) to favor formation of hexameric structures. In anothervariation, there may be two aldehyde/ketone groups on the “bases” whilethe aromatic ring above the “bases” would have two alcohol groups

The 6 “base” hexamer idea is based on enhancing nucleotide base-pairingwith hydrophobic surfaces. Each heterocycle can form up to 6 hydrogenbonds with its neighbors. While these linker elements may not cometogether in the absence of target, once formed in the presence of targetthey may be rather stable.

In a variation of the above, the aromatic heterocycles are based onnaphthalene framework (two fused 6-membered rings) instead of 6 memberrings. The advantage of this design is it creates a larger aromatichexameric surface. Once formed, such a hexameric structure may also helpcondense a head-to-head second hexameric structure onto the firstaromatic face. The resulting super-structure would be composed of 12coferons and could have an affinity to targets that rivals the affinityof antibodies.

In a variation of the above, one could form trimers instead of hexamers.For example, instead of double U, just use two coferons with U that havean aromatic ring containing a single aldehyde, and one coferon with adouble mini-diamino A base and an aromatic ring with two alcohols in themeta positions. This structure may be nicknamed the “half-pipe”. It canalso form head-to-head structures so that 6 coferons are all pointingtoward the same direction.

In other variations, not using hydrogen bonding among underlyingnucleotides, but just focusing on the concept of having “N” coferonmonomers come together in the proper geometry, the aromatic ring ideacan be extended to form pentameric coferon multimers by using 5 memberheterocycles so that 108° geometries can be established.

Derivative Based on Base-Pairing Dimers that Form Multimers

These linker elements are derived from Linker Element 9. In thisembodiment, a layer of 6 bases all pairing and forming 6 hydrogen bondseach in a circular hexagonal planar structure, each with a second layerof 6 bases all pairing and forming 4 hydrogen bonds each that goes abovethe bases, allowing for covalent bonding between an aldehyde/ketone onone side of the aromatic ring with an alcohol on the adjacent ring. Thealdehyde/ketone and alcohol groups are in the ortho orientation on thearomatic ring (120° orientation) to favor formation of hexamericstructures. This is Linker Element 19.

19A)

In a first design boric acid esters form dimers that then form hexamers.In this design, both the top nucleotides and the bottom nucleotides arecovalently linked through a boric acid ester in the ortho positions, butthey do so alternating from top to bottom. Thus, the top nucleotides arelinked from left to right, while the bottom nucleotides are linked rightto left.

A top view of the top and bottom rosettes of the above design shows howall the nucleotides hydrogen bond at every position, and how thecovalent linkages are offset from each other, so that all 6 coferons arecovalently linked.

Top View of the Upper Layer.

Top View of the Bottom Layer.

In a second design that uses boric acid esters to form dimers that formhexamers only the top nucleotides are covalently linked through a boricacid ester in the ortho positions. In this design, both nucleotides aresymmetric along the 1-4 axis, and thus can rotate in either direction.

A top view of the rosette of the above design shows how all thenucleotides hydrogen bond at almost every position, skipping the middlehydrogen bond in every other pair. The advantage is that all 6 coferonsare covalently linked on the top rosette.

Derivatives Based on Forming Tetramers of Nucleotides of1,3-Diaminopyrimidine, Diaminopyridine, or Aminopyrimidine Base Pairswith Cytosine or 5-Amino-2-Pyridone.

These linker elements are also derived from Linker Element 9.

In one embodiment, the two backbones are linked through hemiacetalsafter stacking.

In another embodiment, a linear peptide backbone may be used for thenucleobase linker elements. In such a monomer, the diversity elements ofthe monomer are the side chains of alternating amino-acid residues Inanother embodiment, the backbone that bears the nucleobase linkerelements may be a circular peptide nucleic acid backbone. In such acoferon monomer, the diversity groups are the side chains of adjacentamino acid residues. In yet another embodiment, a linear backbone basedon a cyclopentane scaffold may be used.

The 1,3-diaminopyrimidine, diaminopyridine or aminopyrimidine base pairswith cytosine or 5-amino-2-pyridone tetramer idea is based on enhancingnucleotide base-pairing with hydrophobic surfaces. Each bottomnucleotide can form 3 hydrogen bonds with its pair. Each top nucleotidecan form 2 hydrogen bonds with its pair and is attached through ahemiacetal linker. The top nucleotides can become covalently linkedacross the hydrogen bond area in the “major groove”. This structure cannow be flipped 180° and stacked with its pair so that there are 4 basesstacked onto each other on one discontinuous strand, and an additional 4bases stacked onto each other in a second discontinuous strand. The twosets can be linked either through groups on the bases, or the backbone.The 4 diversity elements emanate from the backbones, and can be thoughtof as coming out of the minor groove of a 4 base double helix. Thisstructure will be very stable once formed, and be able to create abinding surface of the same general size as a single antibody heavy orlight chain. In the case of coferon dimers, the hemiacetals betweenbackbones or between bases are not used.

Linker Elements Based on Forming Heterocycle Dimers Using Boric AcidEsters to Link the Nucleotides.

In this embodiment, covalent bonds are formed on the upper base pairthrough a boric acid ester in the “major groove”. The top heterocycleshave two hydrogen bonds. The lower base pair has either three hydrogenbonds, or two hydrogen bonds and a boric acid ester. This structure cannow flip 180° and stack with it's pair so that there are 4 bases stackedonto each other on one discontinuous strand, and an additional 4 basesstacked onto each other in a second discontinuous strand. The two setscan be linked either through ethyl alcohol and aldehyde groups on thebackbone or through boric acid and alcohol groups on the bases. Thisstructure will have enhanced stability once formed, and be able tocreate a binding surface of the same general size as a single antibodyheavy or light chain.

Derivatives Based on [2.2.1] Bicyclo-Heptanone and [2.2.2]Bicyclo-Octanone Structure (Linker Element 21).

In one variation the ketone is in the 2 position, and the alcohol groupis in the 6 position in the equatorial position (pointing slightlyupward). In another variation an aldehyde or ketone group is attached atthe 1 position, with an alcohol at the 4 position. Such bicycloderivatives would not be able to form two covalent hemiacetals orhemiketals when forming a dimer.

Derivatives Based on Alternate Stacking of Aromatic Rings

Derivatives based an alternate stacking of aromic rings containingalcohol and aldehyde groups, such that the groups cannot reactsimultaneously to form two covalent bonds between two linker elements(Linker Element 22).

In one variation of this theme, one linker element contains twoaldehydes in the meta orientation (1,3), the second contains twoalcohols, but there is a bulky substituent (such as tert-butyl,isopropyl, ethyl, methyl, Br, Cl, I) in-between the two (2 position onthe ring).

R=isopropyl, t-butyl, ethyl, methyl, halogen or other sterically bulkysubstituent.

E=

D=—(CH₂)_(n)OH, n=2-3

where the dashed line crossing the solid line represents the one or morediversity elements attached, directly or through a connector, to thelinker elements.

Thus, when the two aromatic rings stack, they need to be offset by 120°.This type of design will also rotate the position of the diversityelements by 120°, however, this should not influence binding to thetarget if the linker elements are connected to the diversity elementsvia flexible ethylene glycol connectors.

-   22A)

In another variation of this theme, the two linking groups are designedsuch that when one pair covalently link, the other two pairs are too farapart to also react. For example, one linker element aromatic groupcould have two aldehye groups in the ortho orientation, while the otherlinker element would have the alcohol groups in the para orientation.

-   22B)

Alternatively, a naphthalene type structure could be used. When suchlinker elements stack, the aromatic group rotates back and forth, thusthis set is known at the “flip/flop” design.

-   22C)

Derivative Based on Intercalating Aromatic Rings.

Linker elements that feature aromatic rings that are separated by arigid spacer have been described earlier to form dimers and trimers.When the aromatic rings on two such linker elements intercalate, theyreduce the total number of aromatic surfaces exposed to aqueous solutionfrom 8 down to 2. Including aldehyde and/or ketone groups and alcoholgroups on alternating linker elements such that they are offset withrespect to each other, a linker element pair can stack onto of anotherlinker element pair which then become covalently linked to each other.This architecture may be used to construct linear coferon multimers, asshown below.

Derivatives that Combine Metal Binding Group with Covalent BondFormation

Linker elements that combine zinc chelators with hemiacetal covalentbond formation allows assembly of tetramers. This is Linker Element 24.A variation using picolinic acid as the chelator is shown in the examplebelow as 24A).

24A)

Another variation of Zn chelating coferons that form tetramers is basedon binding of two zinc cations and formation of boric acid esters. Thisdesign takes advantage of aromatic ring stacking of the picolinic acidgroups. The ethyl alcohol is perfectly positioned to form a covalentlink to the boric acid on the aromatic of the neighboring picolinic acidlinker element.

Derivatives Based on a Single Linker Element that Assembles to FormTrimers or Hexamers.

These derivatives are based on forming boronate esters between boronicacid and 1,2 and 1,3-diols (Linker Element 25). When derived fromaliphatic molecules, these linker elements form dimers. When derivedfrom an aromatic ring containing molecule, these linker elements canform trimers or hexamers. Boronates can also complex with amino alcoholsand may also complex with amido alcohols.

-   where R is an aliphatic or alicyclic group including —H, —CH₃, —CF₃-   where Q is an aromatic heterocyclic or nonheterocyclic ring-   where n=0-3, m=1-3 and p=1-2    where the lines crossed with a dashed line illustrate the bonds    formed joining the one or more diversity elements to, directly or    through a connector, the molecules of Formula (B1), Formula (B2),    Formula (B3) and Formula (B4).    Examples of these embodiments are shown below as 25A) to 25 D)

In these designs an aromatic ring (that may contain N, S or O atoms atvarious positions within the ring) bears a boronic acid moiety as wellas a 1,3-diol moiety in the meta position of the aromatic ring. Theselinker elements can form trimers as well as hexamers

25A)

In another embodiment, the boronic acid moiety as well as the 1,3 diolmoiety may be replaced by a hydroxymethyl ketone leading to hexamericassembly through the formation of hemiacetal covalent bond formation.

-   25B)

-   25C)

-   25D)

Derivatives that Form Dimers Which Interact with Different Monomers toForm Tetramers

Derivatives based on linker elements that first form dimers throughhemiacetal formation, thus positioning two sets of hydroxyl groups thatare optimally positioned to react with a boronic acid moiety on anaromatic ring (that may contain N, S, or O atoms at any position withinthe ring). This is Linker Element 26.

Derivatives Based on Assembly of Three Separate Linker Elements

Derivatives based on trimeric assembly of linker elements where covalentbond formation between a first linker element and a second linkerelement generates a new reactive group on the second linker elementallowing it to form additional covalent bonds with a a third linkerelement (Linker Element 27). The first linker element may be analiphatic or aromatic amine or imidamide and reacts with a second linkerelement that may be a cyclohexanone bearing a hydroxymethyl substituentat the 2-position. The third linker element may be an aromatic ring(containing N, S, or O atoms at any position within the ring) and aboronic acid moiety.

-   27A)

Example 2

-   27C)

Derivatives Based on Linker Elements that are Actively Transported intoTarget Cells

Linker elements that contain folic acid will preferentially enter cancercells using the folate transporter (Linker Elements 28). In oneembodiment, diversity element is attached to the folic acid based linkerelement via an ethylene glycol linker.

-   28A)

In another embodiment, the two folate moieties have the opportunity tobecome reversibly covalently linked to each other.

-   28B)

In a third embodiment, the folic acid moiety is attached to a secondlinker element via a disulfide bond. Cleavage of the disulfide bond byglutathione within tumor cells now facilitates irreversible binding oftwo linker elements through formation of a thiazolidine ring.

-   28C)

In other variations, the linker element may be attached to othermoieties to make linker element precursors that bind receptors ortransporters that then facilitate transport or uptake of the linkerelement precursors in to the cell.

Derivatives Based on Intracellularly Activated Irreversible CrosslinkingLinker Elements

In these designs, the linker element contains a protected functionalgroup that undergoes deprotection inside the cell. The resultingfunctional group allows for irreversible covalent bond formation betweenthe linker elements (Linker Element 29). One embodiment of this designcontains a protected disulfide. On entering cells the disulfide isreduced by glutathione releasing a free sulphydryl group that is beta toa primary amine. This moiety can then react irreversibly with a carbonylon another coferon monomer to form a thiazolidine linkage.

-   29A)

Such linker elements may be designed to carry both the protecteddisulfide as well as a carbonyl such that on generation of the freesulfhydryl group, the monomer can react with itself to form a homodimer.

-   29B)

An alternative mode of dimerization for the above linker element isshown below

Diversity Elements

Most drugs work by blocking protein activity, clogging an enzymaticpocket, and thus inhibiting activity. In order for a drug to bind, thereneeds to be sufficient complementarity and surface area of contact suchthat van der Waals and ionic interactions provide the requisite bindingenergy. The field of combinatorial chemistry is based on the principleof creating ligands or diversity elements of different shapes and sizes,some of which can bind to the desired surface of the target, and thusserve as lead molecules for subsequent medicinal chemistry.

Coferons have the advantage of being able to bind the target through twoligands or diversity elements. These diversity elements combine to givethe coferon a tighter binding than would be achieved through a singlediversity element. In addition, coferons provide a linker element (andan optional connector), which may provide additional opportunities tomaximize the surface area of interaction between the coferon and proteintarget.

Combinatorial chemistry approaches seek to maximize diversity elements,and such molecules are often synthesized using split and recombine orbead-based approaches. There are two general approaches used to generatea diversity library: (i) a single platform with multiple functionalgroups, each of which is reacted with a family of diversity reagents tocreate a library of surfaces and (ii) the diversity is generated usingbifunctional reagents to create short linear or circular chains, such aspeptides and peptide analogues.

In many of the examples below, the order of synthesis is a linkerelement is attached to a tri-functional connector, with one of thefunctionalities used to attach the connector-linker element to a bead orDNA barcode or tag sequence. This is followed by attaching orcombinatorial synthesis of the diversity library of ligands. The orderof these steps and the geometry of the components may be altered. Forexample, the linker element may also double as the connector, beingattached to the diversity element on one end and the bead on the otherend. Also, the linker element may be added last, after synthesis of thediversity element. The examples below are by no means exhaustive ofmethods for synthesizing linker elements with diversity elements.

Diversity elements may be molecules previously known to bind to targetproteins, molecules that have been discovered to bind to target proteinsafter performing high-throughput screening of previously synthesizedcommercial or non-commercial combinatorial compound libraries ormolecules that are discovered to bind to target proteins by screening ofnewly synthesized combinatorial libraries. Since most pre-existingcombinatorial libraries are limited in the structural space anddiversity that they encompass, newly synthesized combinatorial librarieswill include molecules that are based on a variety of scaffolds.

Monocyclic Scaffolds

These scaffolds may be used to generate the simplest types ofcombinatorial libraries.

Monocyclic scaffolds

In addition to those nitrogen and carbon atoms that are substituted byR₂ and R₃, other positions may contain additional substituents includingH. Multiple bonds may also be incorporated between ring atoms.

Bicyclic Scaffolds

Each bicyclic scaffold may be substituted at different positions andcontain heteroatoms and multiple bonds as illustrated for monocyclicscaffolds above.

Tricyclic Scaffolds

Tricyclic scaffolds containing 3 rings fused to each other and maycontain heteroatoms and multiple bonds as illustrated for monocyclicscaffolds above.

Tetracyclic Scaffolds

Tetracyclic scaffolds containing 4 rings fused to each other and maycontain heteroatoms and multiple bonds as illustrated for monocyclicscaffolds above.

Spiro Scaffolds

Spiro scaffolds where two rings are fused to each other through a singlecommon atom

Multicore Scaffolds

Multicore scaffolds where each of the above scaffold core elements arelinked by a covalent bond.

Connectors

In one embodiment, a linker element is attached to a tri-functionalconnector, with one of the functionalities used to attach theconnector-linker elements to a bead. Beads are distributed to uniquewells, and a set of diversity elements react with the third functionalgroup on the connector (for example 500 different aldehyde containingmoieties reacted with an amino group). In this embodiment, the well thesynthesis took place in identities the diversity element.

In a second embodiment (FIG. 2.1A), a linker element is attached to atri-functional connector, with one of the functionalities used to attachthe connector-linker element to an encoded bead. For example, Veracode™beads (Illumina, San Diego, Calif.) or silicon particles may be used,where each bead has a unique Veracode™ or barcode pattern. The beads orparticles are distributed into a set of reaction chambers (for example10 chambers), identified in each chamber, and then reacted with abifunctional moiety (for example, a protected amino acid). The beads aremixed, split again into the reaction chambers, and the process isrepeated (split-pool synthesis). In this embodiment, repeating theprocess a total of 4 times will result in 10,000 diversity elements inthe library. In a variation of this approach, at the end of thesynthesis, the last amino acid residue is reacted with the connector tocreate a circular diversity element. In this version, the diversityelement is identified by the code on the bead or particle.

In a third embodiment, a linker element is attached to a tri-functionalconnector, with one of the functionalities used to attach theconnector-linker element to a long DNA barcode sequence (FIG. 2.1B). TheDNA barcode has two universal primer binding site sequences at the 3′and 5′ end, and contains multiple internal zip-code sequences, which aresequences that have similar melting temperatures but are different in atleast 25% of their bases so that they uniquely hybridize to theircomplementary sequences either in solution, on beads, or on arraysurfaces. The molecules comprised of connector—linker element—DNAbarcode (=library members) are equilibrated with a set of columns (e.g.,10 columns), containing beads with complementary zip-code sequences. DNAhybridization captures library members containing the complementaryzip-code sequence on their DNA barcode. At the end of this process, allthe library members should be distributed in the 10 columns, dependingon the zipcode sequence in the first position of the DNA barcode. Thelibrary members are eluted into separate new chambers, and reacted witha bifunctional moiety (e.g., a protected amino acid) that corresponds tothe given zip-code. The library members are then re-pooled and thenrerouted to the next series of columns, using the zipcode sequences inthe second position of the DNA barcode for sequence-specific capture. Inthis embodiment, repeating the process a total of 4 times will result in10,000 diversity elements in the library. As above, at the end of thesynthesis, the last amino acid residue may be reacted with the connectorto create a circular diversity element. In this embodiment, the identityof the diversity element is defined by the zip-code sequences in the DNAbarcode.

In a fourth embodiment, a linker element is attached to a tri-functionalconnector, with one of the functionalities used to attach theconnector-linker element to a DNA template sequence containing twouniversal primer binding site sequences at the 3′ and 5′ end, andmultiple zipcode sequences. The DNA sequence serves as a template foradding bifunctional moieties to the remaining functional group on theconnector. Each bifunctional moiety is attached to a complementaryzip-code DNA molecule, which hybridizes on the DNA template containingthe connector-linker element. This hybridization increases the localconcentration of the reactant to such an extent that it can drivesynthesis to very high yields. This method does not requiresplit-pooling techniques. In this embodiment, the first set ofbifunctional moieties are attached to complementary zip-code DNAmolecules to the first set of zipcodes, the second set of bifunctionalmoieties are attached to complementary zip-code DNA molecules to thesecond set of zipcodes, etc. This approach has the disadvantage ofrequiring synthesis of functional moieties attached to every new set ofcomplementary zip-code sequences. Another problem is each additionalround moves the reactive group further away from the growing chain, aproblem which can be ameliorated by using oligos designed to hybridizeover a split sequence like an “omega” symbol. To circumvent the need togenerate new reagents containing bifunctional moieties for each new setof zipcodes, another variation would use three oligonucleotide sets. Thefirst set contains the connector—linker element—DNA template sequencelibrary covalently linked to the growing diversity element. The secondset contains a unique zipcode for each bifunctional moiety used. Thethird oligonucleotide set is complementary to the particular zipcodesequence on the bifunctional moiety from the second set as well as theparticular zipcode in the DNA template sequence from the first set, andby hybridizing to both of these DNA elements, it brings the bifunctionalmoiety in close proximity with growing chain on the connector—linkerelement—DNA template thus increasing the local concentration and drivingthe reaction forward. This method has the advantage that it does notrequire split-pooling techniques. If 4 sets of 10 each bifunctionalmoieties are added, this will result in 10,000 diversity elements in thelibrary. As above, at the end of the synthesis, the last amino acidresidue may be reacted with the connector to create a circular diversityelement. In this version, the identity of the diversity element isdefined by the zipcode sequences in the DNA template.

In a fifth embodiment, a linker element is attached to a tri-functionalconnector, with one of the functionalities used to attach theconnector-linker element to a DNA barcode sequence containing twouniversal primer binding site sequences at the 3′ and 5′ end, and asingle zip-code sequence in the middle. The molecules are equilibratedwith an array surface containing complementary zip-code sequences,positioned such that a multi-chambered cover could divide the array intochambers (e.g., 10 chambers). Each chamber is reacted with a differentbifunctional moiety (e.g., a protected amino acid) that corresponds tothe given zipcode. The library members are then re-pooled and thenrerouted to the next array, where the complementary zip-code sequencesare now rearranged. In this example, repeating the process a total of 4times will result in 10,000 diversity elements in the library. In avariation of this approach, the array complementary zip-code sequencesmay be modified on their free end to become covalently attached to agiven bifunctional moiety. The moieties are first added to the correctzipcodes using the chambers. Subsequently, the chambers are removed, andthe library members containing DNA barcode sequences are added to thearray. Once they hybridize to the correct addresses, they are properlyoriented to react with the bifunctional group on the zipcode and addthat moiety to the growing chain. In a full variation of this approach,the array addresses are printed to contain both the complementaryzip-code sequences and a contiguous DNA sequence tag on the same strand.Under these conditions, both a DNA molecule complementary to the tagsequence and containing a bifunctional moiety, as well as the librarymember with the cognate zip-code sequence, can bind to the address toset up a DNA targeted synthesis step. This full variation would obviatethe need for chambers to separate the individual reactions. As above, atthe end of the synthesis, the last amino acid residue may be reactedwith the connector to create a circular diversity element. In thisversion, the identity of the diversity element is defined by the singlezipcode sequence in the DNA barcode.

In a sixth embodiment, a linker element is attached to a tri-functionalconnector, with one of the functionalities used to attach theconnector-linker element to either a Veracode™ bead, bar code particle,or a DNA barcode sequence containing one or more zipcode sequences. Theremaining functionality is connected to a “platform” containingadditional functionalities. For example, the platform may be acyclo-pentane derivatized on three carbons all in the syn orientation.In this version, one of the encoding processes described in embodiments2-5 above is used to add mono-functional moieties to the appropriatefunctional groups on the platform. For example, if there are 20 moietiesadded in each step, the resultant library will contain 8,000 diversityelements. The advantage of this approach is to guide all the diversitycomponents in a single orientation for maximum diversity in bindingsurfaces.

Encryption Portion

The most versatile encryption element is an attached bar code, typicallya nucleic acid sequence, such as an oligonucleotide or DNA. Theencryption element may be used to directly assist in synthesizing thediversity element (as in DNA Targeted Synthesis (“DTS”)). Eachencryption element is composed of multiple zipcode sequences. Theencryption element has a chemical molecule attached to one end. Uponhybridization of a first zip-code complement bearing a reactant groupwith a diversity element, the reactant group is covalently linked to,and transferred to the chemical molecule on the nucleic acid. Eachunique zipcode complement is charged with a reactant group containing adiversity element that corresponds to the zipcode sequence. The processis repeated with a second set of charged zipcode complementscorresponding to the second zipcode in the encryption element sequence.At the end of several rounds of synthesis, adding diversity elements ateach step, there is the optional step of circularizing the chain.Alternatively, the encryption element may be used to capturing certainproducts to defined columns. In this embodiment, each encryption elementis also composed of multiple zipcode sequences. The sequences are passedover columns containing zip-code complements to the first set of zipcodesequences. The process is repeated until the nucleic acid sequences arebound to the proper column containing the correct zip-code complements.Subsequently, each nucleic acid column is eluted into a separatereaction chamber, whereupon the corresponding diversity element ischemically reacted to a chemical molecule attached to the encryptionelement. The process is repeated with a second set of columns containingzipcode complements corresponding to the second zipcode in theencryption element sequence. At the end of several rounds of synthesis,adding diversity elements at each step, there is the optional step ofcircularizing the chain. In another embodiment, the encryption elementmay be captured on beads, or addresses on solid supports for asubsequent round of synthesis, or used for zip-code identification ofthe final ligand. The finally selected ligands may be identified by PCRamplification, followed by sequencing the zip-code portions of theamplicons. Individually amplified molecules are captured onto beads orsolid supports and amplified such that all the molecules on anindividual bead or cluster on a solid support are identical. Thesequence of each bead or cluster is then determined using sequencing bysynthesis or pyrosequencing, or sequencing by ligation. The zipcodesequences on each bead or cluster may also be determined by hybridizingfluorescently labeled pooled zipcode complements, and scoring which setgave a signal, creating a binary code to finally identify uniquezipcodes at defined positions. Alternatively, zip-codes may beidentified by hybridizing onto an array.

An alternative approach, termed RADE (Random Access DNA Encryption)herein, is to randomly encrypt beads particles, or a solid support witha unique sequence, replicate multiple copies of that uniqueoligonucleotide sequence on the bead, and then leave a copy (footprint)of the complement of that unique sequence wherever that bead has gone.This marks where the bead was when a given diversity monomer was addedto synthesize the complete ligand. Once the winning ligand(s) has/havebeen selected, PCR amplification followed by sequencing identifies theunique sequence(s) associated with the ligand(s). Unique PCR primers aredesigned to the unique regions of each sequence, and then may be used tointerrogate wells to determine the tracks of each step in the synthesis.

There are several approaches for generating beads or particles withmultiple copies of the same sequence on one bead, but with each beadcarrying a unique sequence. To facilitate such attachment ofoligonucleotides to the beads or solid support, the beads or solidsupport are functionalized on a fraction of its available surface.Subsequently, coferons are synthesized on either the bead, or on the endof the oligonucleotide barcode. In a preferred variation, the coferonsare attached to the bead or oligonucleotide via a photolabile linker. Afew of the approaches for generating such constructs are listed below:

-   A. Goal: Generate beads with hundreds to thousands of copies of the    sequence (below) across the entire surface of the bead (See FIG. 3).-   UniA (20)-Rand (20)-UniB (15)-Rand (20)-UniC (20)-   Where:-   UniA is universal or common sequence A, 20 mer.-   Rand is a randomly generated or unique 20 mer sequence.-   UniB is universal or common sequence B, 15 mer.-   Rand is a randomly generated or unique 20 mer sequence.-   UniC is universal or common sequence C, 20 mer.-   Step 1A. Synthesize oligos of the following format.-   Primer Seq. 1=Universal 20mer A-Random 20mer-Universal 15mer B-   Primer Seq. 2=Universal 15mer B complement-Random 20mer-Universal    20mer C-   Step 2A. Mix two primer sets together, and add universal primers    (20mers) whose 3′ termini end in A and C, respectively, these will    assemble during PCR amplification to generate 95 base sequences of    the form:-   UniA (20)-Rand (20)-UniB (15)-Rand (20)-UniC (20)-   Step 3A. Prepare emulsion beads and PCR amplify so that each bead    has several copies of the same 95 base sequence. In emulsion PCR,    one primer (A) is attached to the bead, the 95 base sequence is    captured at less than one per bead, and then a lower concentration    of primer A and a higher concentration of primer C, Taq polymerase,    dNTPs, Mg are added, emulsified around the beads and then generate    copies of the same fragment on each bead as individual PCR chambers    (Frank Diehl et al, Proc. Nat'l Acad Sci USA, 102:16368-16373    (2005), which is hereby incorporated by reference in its entirety).-   Step 4A. Coferons may be synthesized on activated groups directly on    the surface of the bead, or in a variation, a primer containing a    functional group is ligated to the end of the amplified product.    Thus, the coferon will be synthesized at the end of the DNA strand.    This presents two opportunities: (a) with the coferons at the end of    the DNA, they are in easy reach of the protein target, and (b) this    design provides the option of either cleaving the coferon off the    end of the bead, or cleaving the DNA off the bead, and then it is    still attached to the coferon.-   B. Goal: Generate beads with hundreds to thousands of copies of the    sequence (above) across one face of the bead or particle. (See FIG.    4).-   Step 1B-28. Same as above.-   Step 3B. Activate only one side or surface for attaching the A    primer. This may be done by having the beads float on the surface of    some liquid, and spraying an activating chemical on only the exposed    surface. Another approach is to print the activating group on the    surface of a silicon or glass wafer, and etch away horizontal and    vertical sections, leaving millions of particles that are just    etched on one side.-   Step 4B. Prepare emulsion PCR as above. The advantage of limiting    the PCR product to one face of the bead is to avoid having too many    DNA molecules sticking out, that may later interfere with the    coferon synthesis or binding of fluorescently labeled target    (protein) to the bead.-   C. Goal: Generate beads with hundreds of tandem-repeat copies of the    sequence (below) across one face of the bead or particle. (See FIG.    5).-   UniA (20)-Rand (20)-UniB (15)-Rand (20)-UniC (20)-UniD (20)-   Step 1C. Synthesize oligos of the following format.-   Primer Seq. 3=5′ blocked-T5-Universal 20mer D-Universal 20mer    A-Random 20mer-Universal 15mer B-   Primer Seq. 4=5′ p-Universal 15mer B complement-Random    20mer-Universal 20mer C-Universal 20mer D complement--   Step 2C. Mix two primer sets together, they will hybridize at the    two complementary regions to form a 115 base circle which is gapped    on both strands. Add DNA polymerase lacking 5′-3′ or strand    displacement activity (such as Klenow), dNTPs, ATP, and T4 DNA    ligase. The polymerase will extend both free 3′ ends, but only    ligate the Primer Seq. 4 strand to form a circle. The other strand    cannot ligate because the 5′ end is blocked. By subsequently adding    a strand displacement polymerase (such as Bst Polymerase or Phi-29    polymerase), hundreds of tandem-repeat copies of the sequence below    may be generated using rolling circle amplification.-   UniA (20)-Rand (20)-UniB (15)-Rand (20)-UniC (20)-UniD (20)-   Step 3C. Using beads or particles with only one side or spot    activated and containing the UniD complement sequence, capture the    long single-stranded DNA, with one DNA multimer going per bead or    particle. The original 5′ end (synthesized Primer seq. 3) may have    been modified allowing for covalent capture to the bead or particle.    The strand will be captured at a number of positions along the    length of the single strand. The UniD complement capture sequence    may be composed of PNA, or 2′ O-methyl containing sequences, such    that this DNA stays hybridized even under conditions where copies of    the UniA (20)-Rand (20)-UniB (15)-Rand (20)-UniC (20) sequence are    generated, and then melted off the beads, the easiest being by using    a helicase that does not recognize and unwind the PNA, or 2′    O-methyl containing sequences.

Once beads or particles have been generated, they may be used tosynthesize the coferons. For the purposes of this example, the coferonsynthesis has three rounds of diversity, of 20 different groups, andthere are 20 different scaffolds (160,000 fold diversity). Aftersynthesis of the linker elements and connector portions, the beads aredivided into 20 wells and the first round of diversity elements areadded. Afterwards, a universal primer is added, hybridizing to all theDNA fragments, extended, and then melted off (by either heat or high pH,organic solvent such as formamide, or a helicase). After removal of thebeads, the 20 wells contain a footprint of all the beads in those wells.

The beads from the 20 wells are then pooled and split into a new 20wells. The process is repeated 3 more times, leaving 80 wells withfootprints.

The beads are used in a selection (with the other coferon library addedin solution) to bind the fluorescently labeled target. The winning beadsare separated, and the DNA barcodes PCR amplified. The amplifiedfragments are sequenced using solid phase techniques (Next-Gensequencing). Note also, that the same approach may be used withindividual coferons that still contain the DNA tag attached to it.

The sequences are recorded, and forward and reverse primers aresynthesized within the unique 20 mer sequences. There is someflexibility in the exact region used to synthesize the unique primer,for example it may use 14 to 15 bases of the random sequence and 5 basesof the UniA and UniC sequences, respectively. This flexibility allowsthe user to avoid using a primer that ends in a palindromic sequencewhich will most likely make a primer dimer.

Using the unique primer pairs synthesized for each winning sequence, theoriginal 80 wells (a single run on a 96-well real-time PCR machine) areanalyzed via PCR using the middle UniB sequence for detection with aTaqman probe. One well from each set of 20 wells should provide apositive result allowing the user to retrace the tracks of that beadduring each synthesis round, i.e. it's synthesis history.

In practice, one may keep the final 20 wells split, such that each wellcontains 8,000 fold diversity, then when the winning ligand and itssequence are identified, only 60 PCR reactions are required. Likewise,if in the final 2 steps the separation of reactions is maintained=400wells, then each well will contain a 400-fold diversity, andde-convolution requires just 40 PCR reactions.

Alternative means of identifying the diversity element include usingsynthetic steps which incorporate chemical moieties of different mass(for example, amino acid derivatives), and determining the molecularweight of the final product via mass spectroscopy. In addition,diversity elements may be defined by spatial separation, i.e.synthesizing different variants in different wells or chambers. Further,diversity elements may be encoded within the bead or glass surface usedas the solid support for synthesis of ligand. For example, a bead may beencoded with a digital holographic image that may be illuminated by alaser beam and read by a CCD camera (Illumina Veracode™ system).Alternative barcoding schemes include gold/silver nanoparticles, barcoded silicon particles, or using different ratios of embedded quantumdot colors.

Linker Screening

Another embodiment of the present invention involves a method ofscreening for linker elements capable of binding to one another. Thismethod includes providing a first and a second set of monomers. Each ofthe monomers in the first set comprise a linker element, having amolecular weight of less than 500 daltons and being capable of forming areversible covalent bond or non-covalent interaction with a bindingpartner of said linker element, with or without a co-factor, underphysiological conditions and a sulfhydryl group. The linker element andthe sulfhydryl group for each monomer of the first set of monomers arecoupled together. Each of the monomers in the second set comprise alinker element capable of forming a reversible covalent bond orreversible non-covalent bonds with a binding partner of the linkerelement under physiological conditions, an encoded bead, and asulfhydryl group. The linker element, the encoded bead, and thesulfhydryl group for each monomer of the second set of monomers arecoupled together. The first and second sets of monomers are contactedwith one another under physiological conditions so that monomers fromthe first set of monomers and monomers from the second set of monomersbind together to form multimers linked together by disulfide bondsformed from their sulfhydryl groups and, potentially, covalent bonds ornon-covalent interactions between their linker elements. The dimerswhere the linker elements from the monomers of the first and second setsof monomers are covalently bound or non-covalently linked together arethen identified as being candidate multimers. The linker elements fromthe first and second monomers that are covalently bound ornon-covalently joined together are then identified in the candidatemultimers.

The step of contacting is carried out by cycling between conditionsresulting in a high dissociation constant between said linker elementsto allow for re-association of linker elements connected to differentdiversity elements and conditions resulting in a low dissociationconstant between said linker elements to allow for preferential bindingof monomers with the highest affinity diversity elements to the target.

The step of cycling of conditions is achieved by lowering and raisingthe pH or zinc concentration using a membrane permeable to cations andwater.

The identifying dimers step is carried out by determining which dimershave first and second monomers that are more tightly bound together.This is determined by identifying the barcodes of the beads that containlabeled monomers from the first set. In the preferred embodiment, thelabel is a fluorescent group, and the most fluorescently labeled beadsare identified, e.g. in a flow sorter, and separated for identifying thebarcode. If the experiment is performed using the same set of linkerelements both on the bead and in solution, then there will be at leasttwo beads with different barcodes that are fluorescently labeled foreach pair. Monomers are synthesized individually and tested for eachcombination. If the linker elements are different between the bead andin solution, the experiment is repeated a second time, however, in thesecond case the linker elements on the beads and in solution are nowreversed.

In one embodiment of carrying out the identifying step, an encryptionelement comprising terminal universal primer binding sites can beprovided. In using an encryption element, the therapeutic multimerprecursor is contacted with universal primers to form an amplificationmixture. The amplification mixture is subjected to a polymerase chainreaction to form amplification products. The amplification products areidentified and the amplification products are correlated to theoligonucleotides of the monomers forming the therapeutic multimerprecursor. The steps of providing a plurality of monomers, contactingthe plurality of monomers, subjecting monomers to reaction conditions,and identifying the monomers are repeated to determine which of thetherapeutic multimer precursors have a suitable binding affinity to thetarget molecule.

For the monomers with the identified linker elements, the steps ofproviding a first set of monomers, providing a second set of monomers,identifying dimers, and identifying, in the candidate dimers, the linkerelements are repeated. This permits a determination of which of thelinker elements from the first and second sets of monomers have asuitable binding affinity.

The first and second set of monomers may further include an encodingelement, where the diversity element, the linker element, and theencoding element are coupled together. The encoding element can be anoligonucleotide or a labeled bead.

The effectiveness of the linker elements in binding together can bedetermined by a screening method, as described in FIGS. 14A-B.

In one embodiment (FIG. 14A), a library of low molecular weight linkerelements are synthesized on beads which are individually identifiedthrough bar codes. A second library of linker elements, which contains afluorescent label, is synthesized. Different combinations of linkerelements can undergo “dynamic combinatorial chemistry”—i.e. they areassociating and dissociating with each other. Some combinations willbind tighter than others, directing the evolution of combinations to thetightest pairs. For symmetrical libraries, a pair of beads will bedetected for each monomer. The monomers can be resynthesized and testedindividually to find matched pairs.

In the embodiment of FIG. 14B, a library of low molecular weight linkerelements is synthesized on beads which may be individually identifiedwith bar codes. A second library of linker elements, which arecovalently linked to a DNA bar code, are step-wise synthesized.Different combinations of linker elements can undergo “dynamiccombinatorial chemistry”—i.e. they are associating and dissociating witheach other. Some combinations will bind tighter than others, directingthe evolution of combinations to the tightest pairs. The DNA bar codescan be amplified using universal primers to identify individual linkerelements.

In the embodiment of FIG. 15A, a library of low MW (approx. under 300)linker elements is synthesized on beads which may be individuallyidentified through barcodes. A second library of linker elements issynthesized containing a fluorescent label. The bead library linkerscontain tethered disulfide groups, while the solution library linkerscontain tethered sulfhydryl groups. Under incubation conditions,solution linker elements can undergo disulfide exchange with bead linkerelements. Some combinations will bind tighter than others, directing theevolution of combinations to the tightest pairs. For symmetricallibraries, a pair of beads will light up for each monomer. Monomers areresynthesized and tested individually to find the matched pairs.

A library of low MW (approx. under 300) linker elements is synthesizedon beads which may be individually identified through barcodes, as shownin the embodiment of FIG. 15B. A second library of linker elements issynthesized, each monomer covalently linked to a DNA barcode (allowingfor stepwise synthesis and identification of the binding ligand). Underincubation conditions, solution linker elements can undergo disulfideexchange with bead linker elements. Some combinations will bind tighterthan others, directing the evolution of combinations to the tightestpairs.

Monomer Library Synthesis

As shown in FIGS. 16A-C, monomer coferon synthesis is facilitated byusing an encryption element. The most versatile encryption element is anattached nucleic acid sequence, such as DNA. The DNA encryption elementmay be used to directly assist in synthesizing the diversity element.See FIG. 16A. Each DNA encryption element is composed of multiple DNAbarcode sequences, indicated as colored bars. The DNA encryption elementhas a chemical molecule attached to one end. Upon hybridization of afirst barcode complement bearing a reactant group with a diversityelement, the reactant group is covalently linked to, and transferred tothe chemical molecule on the DNA encryption element. Each unique barcodecomplement is charged with a reactant group containing a diversityelement that corresponds to the barcode sequence. The process isrepeated with a second set of charged barcode complements correspondingto the second barcode in the DNA encryption element sequence. In theschematic diagram of FIG. 16A, the coferon monomer on the left showsbarcodes in blue, purple, red, and yellow, with corresponding diversityelements of a blue oval, purple hexagon, red square, and yellow star.The coferon monomer on the right shows barcodes in green, red, yellow,and pink with corresponding diversity elements of a green circle, redstar, yellow hexagon, and pink oval. At the end of several rounds ofsynthesis, adding diversity elements at each step, there is the optionalstep of circularizing the chain. Alternatively, the DNA encryptionelement may be used to capture certain products to defined columns (SeeFIG. 16B). In this embodiment, each DNA encryption element is alsocomposed of multiple barcode sequences. The sequences are passed overcolumns containing barcode complements to the first set of barcodesequences. The process is repeated until the DNA sequences are bound tothe proper column containing the correct barcode complements.Subsequently, each DNA column is eluted into a separate reactionchamber, whereupon the corresponding diversity element is chemicallyreacted to a chemical molecule attached to the DNA encryption element.The process is repeated with a second set of columns containing zipcodecomplements corresponding to the second zipcode in the DNA encryptionelement sequence. In the schematic diagram of FIG. 16B, the coferonmonomer on the left shows barcodes in blue, purple, red, and yellow,with corresponding diversity elements of a blue oval, purple hexagon,red square, and yellow star. The coferon monomer on the right showsbarcodes in green, red, yellow, and pink with corresponding diversityelements of a green circle, red star, yellow hexagon, and pink oval. Atthe end of several rounds of synthesis, adding diversity elements ateach step, there is the optional step of circularizing the chain. Inanother embodiment, the DNA encryption element may be captured on beads,or addresses on solid supports for a subsequent round of synthesis, orused for zip-code identification of the final ligand (FIG. 16C). In theschematic diagram of FIG. 16C, the coferon monomer on the left shows asingle barcode in green, with diversity elements of a blue oval, purplehexagon, red square, and yellow star. The coferon monomer on the rightshows a single barcode in purple with diversity elements of a greencircle, red star, yellow hexagon, and pink oval.

FIGS. 17A-C show monomer coferon synthesis using encoded beads. In FIG.17A, a series of different known inhibitors or analogue ligands to thetarget protein are chemically attached to beads. For example, if thereare 100 inhibitors, the beads are split into 100 reaction vessels. Theinhibitors or analogue ligands added to a given individual bead isdetermined by reading the barcodes of each bead in each of the 100 sets,either before or after the reaction. In FIG. 17A, two inhibitors areindicated by orange and yellow hexagons. A given bead contains only asingle set of inhibitors or analogue ligands. In FIG. 17B, a series ofdiversity elements are added to a common platform. In the schematicdiagram of FIG. 17B, the common platform is indicated by a red triangle.In this process, the beads are split into (n) reaction vessels. Adiversity element is chemically reacted to the common platform attachedto the bead. The diversity element added to a given individual bead isdetermined by reading the barcodes of each bead in each of the (n) sets,either before or after the reaction. The products are pooled, and thensplit again into (n) reaction vessels and the process is repeated.Different reactant groups and protecting groups can guide addition ofsubsequent diversity elements either onto the platform or onto earlieradded diversity elements. In the schematic diagram of FIG. 17B, thecoferon monomers on the bead to the left has diversity elements of agreen oval, blue circle, pink star, and yellow square. The coferonmonomers on the bead to the right has diversity elements of a orangesquare, blue hexagon, pink oval, and yellow star. In FIG. 17C, a secondexample of using a common platform is shown, in this case a cyclopentanescaffold schematically shown as a red pentagon. In this process, thebeads are split into (n) reaction vessels. A first bifunctionaldiversity element is chemically reacted to the linker element on thebead. The diversity element added to a given individual bead isdetermined by reading the barcodes of each bead in each of the (n) sets,either before or after the reaction. Next, the cyclopentane scaffold isadded to the first diversity element. A second diversity element ischemically reacted to the cyclopentane scaffold attached to the bead.The diversity element added to a given individual bead is determined byreading the barcodes of each bead in each of the (n) sets, either beforeor after the reaction. The products are pooled, and then split againinto (n) reaction vessels and the process is repeated. At the end of twoor more rounds of synthesis, adding diversity elements at each step,there is the optional step of circularizing bifunctional diversityelements. In the schematic diagram of FIG. 17C, the coferon monomers onthe bead to the left has diversity elements of a pink star attached tothe linker element and the cyclopentane scaffold (red pentagon), whichsubsequently has attached the diversity elements of a green oval andyellow square. The coferon monomers on the bead to the right hasdiversity elements of a yellow star attached to the linker element andthe cyclopentane scaffold (red pentagon), which subsequently hasattached the diversity elements of blue hexagon and pink oval.

Target Screening

Yet a further embodiment of the present invention is directed to amethod of screening for therapeutic compound precursors which bind to atarget molecule associated with a condition. This method includesproviding a plurality of monomers. Each monomer comprises a diversityelement which potentially binds to a target molecule with a dissociationconstant less than 300 μM and a linker element capable of forming areversible covalent bond or non-covalent interaction with a bindingpartner of the linker element with or without a co-factor underphysiological conditions. The linker has a molecular weight of less than500 daltons. The diversity element and said linker element of eachmonomer are joined together directly or indirectly through a connector.The plurality of monomers are contacted with the target molecule underconditions effective to permit diversity elements able to bind to thetarget molecule to undergo such binding. The monomers are then subjectedto reaction conditions effective for the linker elements of differentmonomers to undergo covalent bonding or non-covalent interactions toform therapeutic multimer precursors, either before, after, or duringthe contacting step. The monomers forming each therapeutic multimerprecursor are then identified.

The step of identifying the monomers can be carried out by determiningwhich therapeutic dimer precursors are more tightly bound to the targetmolecule. This is determined by identifying either DNA barcodes or beadbarcodes. For DNA barcodes, the finally selected ligands may beidentified by PCR amplification, followed by sequencing the barcodeportions of the amplicons. Individually amplified molecules are capturedonto beads or solid supports and amplified such that all the moleculeson an individual bead or cluster on a solid support are identical. Thesequence of each bead or cluster is then determined using sequencing bysynthesis or pyrosequencing, or sequencing by ligation. The barcodesequences on each bead or cluster may also be determined by hybridizingfluorescently labeled pooled barcode complements, and scoring which setgave a signal, creating a binary code to finally identify uniquebarcodes at defined positions. Alternatively, barcodes may be identifiedby hybridizing onto an array.

When each monomer includes an encoding element coupled to the diversityelement and the linker element for each monomer, the identifying step iscarried out by detecting the encoding element in the therapeutic dimerprecursor.

When the encoding element is an oligonucleotide, the oligonucleotide cancomprise terminal universal primer binding sites. The identifying stepcan then include contacting the therapeutic dimer precursor withuniversal primers to form an amplification mixture. The amplificationmixture is then subjected to a polymerase chain reaction to formamplification products. The amplification products are identified andcorrelated to the oligonucleotides of the monomers forming thetherapeutic dimer precursor. The steps of providing a plurality ofmonomers, contacting the plurality of monomers, subjecting monomerswhose diversity elements are bound to the target molecule, andidentifying the monomers can be repeated to determine which of thetherapeutic dimer precursors have a suitable binding affinity to thetarget molecule.

When the encoding element is a labeled bead, the steps of providing aplurality of monomers, contacting, subjecting, and identifying themonomers can be repeated to determine which of the therapeutic dimerprecursors have a suitable binding affinity to the target molecule.

The therapeutic dimer resulting from the above method can be prepared bycoupling the monomers resulting from the identifying step. Subjects withthe condition are identified and the therapeutic dimer are administeredto the selected subjects under conditions effective to treat thecondition.

Therapeutic monomers resulting from the above method can be prepared byproviding the monomers resulting from the identifying step. Subjectswith the condition are selected and the therapeutic monomers areadministered to the selected subjects under conditions effective totreat the condition.

Another aspect of the present invention relates to a therapeuticmultimer precursor. The therapeutic multimer precursor includes aplurality of covalently or non-covalently linked monomers. Each monomercomprises a diversity element which potentially binds to a targetmolecule with a dissociation constant less than 300 μM, a linkerelement, and an encoding element. The linker element has a molecularweight less than 500 daltons and is capable of forming a reversiblecovalent bond or non-covalent interaction with a binding partner of saidlinker element with a dissociation constant less than 300 μM, with orwithout a co-factor, under physiological conditions. The diversityelement and the linker, for each monomer are connected together,directly or indirectly through a connector, and the plurality ofmonomers are covalently bonded together or non-covalently linkedtogether through their linker elements. The diversity elements for theplurality of monomers bind to proximate locations of the targetmolecule.

The libraries described above are in two formats: (i) on a bead or solidsupport with diversity element defined by position or Veracode™encryption of particle, and (ii) off the bead with diversity elementdefined by an encoded DNA element. The advantage of working with coferonlibraries attached to beads is that each bead contains multiple copiesof the identical ligand. This property helps identify the strongestaffinity ligand combinations by the intensity of fluorescently labeledentity captured (i.e. protein or other ligand). The advantage of workingwith coferon libraries encoded by DNA is that selected coferons may beamplified by using their DNA to template a second round of diversityelement synthesis. This allows for evolutionary principles to be used inselecting the best coferons. Further, recent advances in massivelyparallel sequencing technology, such as the Roche 454 andSolexa/Illumina sequencers, allow for sequencing of hundreds ofthousands to millions of DNA products in a single run. See FIGS. 18A-B.Likewise, use of individually encoded beads also allows for directedevolutionary principles to be used in selecting the best coferons. Afterthe winning combinations are identified through the bead barcode, theycan be resynthesized with slight variation in a new round ofsynthesis—followed by a second round of selection. Here, one needs toidentify the chemical structure of both coferon monomers which form adimer. In FIG. 18A, schematic diagrams were presented showing theselection process where one or both elements contained DNA encryption.In FIG. 18B, schematic diagrams are presented where the experiment isrepeated so that the diversity elements from each half of the coferonmay be identified.

FIG. 18A is a schematic overview of directed evolution selection ofcoferons, and their use inside the body. Each coferon monomer includes abinding ligand (diversity element) covalently linked to a DNA barcode aswell as a low MW linker element (dynamic combinatorial chemistryelement), which allow different combinations of ligands to reversiblyassociate with each other. As shown in step 1, when coferon monomers arebrought in contact with the protein target on a solid support, somecombinations will bind tighter than others and, consequently, areenriched. Unbound coferon monomers may be washed away. The most tightlybound pair(s) may be both identified and amplified through the DNAbarcodes. Repeating this process of synthesis-selection-amplificationmimics Darwinian evolution. In a variation of the above approach, asshown in step 2, one coferon monomer includes a binding ligand(diversity element) covalently linked to a DNA barcode as well as a lowMW linker element (dynamic combinatorial chemistry element), while theother is linked to a coded bead. The linker elements allow differentcombinations of ligands to reversibly associate with each other. Whenthe combination of solid-phase and solution coferon monomers are broughtin contact with a labeled protein target, some combinations will bindtighter than others and, consequently, are enriched. The winning pairwill cause that bead to be highly labeled, and this may be isolated byflow cytometry or other methods, and the code identified. The partnersolution phase coferon monomer may be both identified and amplifiedthrough the DNA barcodes. Repeating this process ofsynthesis-selection-amplification mimics Darwinian evolution. The bestcoferon monomers are resynthesized without DNA barcodes for use asorally active drugs, as shown in step 3. Once ingested coferons are in adynamic equilibrium between the monomer form (which can traverse thecell membrane) and the dimer form (which binds to and inhibits theprotein target).

FIG. 18B is a schematic overview of directed evolution selection ofcoferons using only bead encryption. As shown in step 1, a first set ofcoferon monomers comprises a binding ligand (diversity element)covalently linked to a bead containing a unique barcode as well as a lowMW linker element (dynamic combinatorial chemistry element), while asecond set is free in solution. The linker elements allow differentcombinations of ligands to reversably associate with each other. Whenthe combination of solid-phase and solution coferon monomers are broughtin contact with a labeled protein target, some combinations will bindtighter than others and, consequently, are enriched. The winning pairwill cause that bead to be highly labeled, and this may be isolated byflow cytometry or other methods, and the barcode identified. In acompanion selection, as shown in step 2, the second set of coferonmonomers is linked to unique encoded beads, while the first set is freein solution. The linker elements allow different combinations of ligandsto reversibly associate with each other. When the combination ofsolid-phase and solution coferons are brought in contact with a labeledprotein target, some combinations will bind tighter than others, andconsequently are enriched. The winning pair will cause that bead to behighly labeled, and this may be isolated by flow cytometry or othermethods, and the barcode identified. The diversity elements for bothsides of the coferon may be decoded, and then resynthesized withadditional variation. Repeating this process ofsynthesis-selection-amplification mimics Darwinian evolution. The bestcoferon monomers are resynthesized without the encoded beads for use asorally active drugs, as shown in step 3. Once ingested coferons are in adynamic equilibrium between the monomer form (which can traverse thecell membrane), and the dimer form (which binds to and inhibits theprotein target).

FIG. 18C is a generic summation of screening for the tightest bindingcoferons using directed evolutionary principles. Individual coferons, ormultiple copies of the identical coferon on individual beads orparticles, or multiple copies of identical coferons within encodeddroplets may be screened by a number of different assays that identifybinding diversity elements. The nature of these diversity elements isdetermined by identifying the code that corresponds to the diversityelement, which is then resynthesized, including minor variations. Theprocess is repeated until the tightest binding elements are identified.

The best coferon monomers are resynthesized without DNA barcodes orencoded beads for use as orally active drugs. The coferons may beprovided as (i) therapeutic dimers or multimers thatdissociate/re-associate in the body, cell, or cellular compartment, (ii)therapeutic monomers in the same or different pills, or (iii)therapeutic monomer precursors where one or more active moieties is in aprotected state, suitable for deprotection once inside the body, cell,or cellular compartment. Once ingested, coferons are in a dynamicequilibrium between the monomer form (which can traverse the cellmembranes), and the dimer or multimer form (which binds to and inhibitsthe protein target).

To recapitulate the opportunities presented by dynamic combinatorialchemistry, FIG. 19 illustrates an idealized case. Different combinationsof linker elements may associate with each other. However, since theinteraction between the linker elements is both weak and reversible, themolecules are in constant flux between the monomer and dimer forms. Whena target is present, it will bind the winning pair of coferons tighterthan other pairs. In doing so, it removes the winning pair fromsolution. Dimer coferons containing one of the winning pair diversityelements will now dissociate, and the resultant monomers reassociatewith each other to bring the concentration of the winning pair back intoequilibrium. However, now that new winning pair also gets bound by thetarget. The process repeats itself and drives concentration of thewinning pair directly on the target. Dynamic combinatorial chemistryalso works when the majority of coferons are monomers, and the diversityelements bind to the target individually as monomers, wherein the targetliterally acts as a catalyst to accelerate formation of its owninhibitor (FIGS. 40-47).

FIGS. 2.1C-2.1D show dimers resulting from screening coferon monomerswith connectors, while FIGS. 2.1H-2.1I show dimers derived from a screenwith coferon monomers which are not provided with connectors.

Each coferon monomer consists of a binding ligand (diversity element)covalently linked to a DNA barcode as well as a low MW linker element(dynamic combinatorial chemistry element), which allow differentcombinations of ligands to reversibly associate with each other. Whencoferons are brought in contact with the protein target on a solidsupport, some combinations will bind tighter than others and,consequently, are enriched. Unbound coferons may be washed away. Themost tightly bound pair(s) may be both identified and amplified throughthe DNA barcodes. Repeating this process ofsynthesis-selection-amplification mimics Darwinian evolution.

Under physiological conditions, different combinations of ligands areforming and reassociating with each other. The term “physiologicalconditions” is hereby defined as aqueous conditions inside the body orthe cell, comprising a temperature range of about 35-40° C., a pH rangeof about 6-8, a glucose concentration range of about 1-20 mM, and anionic strength range of about 110 mM to about 260 mM.

When the diversity elements are brought in contact with the proteintarget on a surface, some combinations will bind tighter than others.This directs the evolution of combinations to the preferred pairs. Afterremoving unbound ligands. DNA encoding regions (colored lines,“zip-codes”) may be amplified using universal primers (black lines) toidentify individual ligands, which serve as lead molecules.

In the examples below, the selected binding elements are referred to asthe top set of coferons. This refers to the entire molecule, from thelinker element to the connector to the diversity element ligand. In manycases, this also includes the encoding DNA template or barcode sequence.Appropriate controls are needed to assure that the monomeric linkerelement portion or the single-stranded DNA portion is not skewing thebinding or selection process. One approach to nullify such effects is toadd the partner linker element, with no diversity element attached. Asecond approach is to hybridize complementary universal PCR primer andsynthesize the opposite strand of the DNA encoded element to make itdouble stranded.

As shown in FIG. 20, a first version of a screen uses diversity elementson beads. Fluorescently labeled target protein is added to the beadsand, after a suitable period of incubation under conditions of gentlemotion (known as panning), wells containing fluorescently labeled beadsare identified. If the beads are in a single chamber, the Veracode™ isidentified for the fluorescently labeled beads. This approach identifiesthe diversity elements that bind most tightly to the target (withaffinities in the micromolar to nanomolar range). The top set of thesecoferons (for example, the top 100) are then re-synthesized ontoindividual beads. In a variation of this theme, each of these topligands can be attached through a series of connectors that vary insize, flexibility, or for circular diversity elements, the size of themacrocycle. This new set of coferons are now combined with a library ofcoferons in solution. It can be the same set of diversity elements asabove, but now instead of being on a bead they have been released insolution. Alternatively, they may have been synthesized with a DNA tagor identification sequence. Now, addition of the fluorescently labeledtarget protein to the coferon containing beads and the coferons insolution at the appropriate concentrations will allow for selection ofthe tightest binding combinations. The bound coferons are in equilibriumwith the coferons in solution, both binding and coming apart. Meanwhile,the protein targets are binding and dissociating with coferons insolution and on the solid supports. The most stable complexes of beadcoferon to solution coferon to target protein are removed from thisequilibrium. The concentration of these components in solution has nowdecreased, so they dissociate from less stable complexes. This nowdrives the equilibrium towards forming even more of the most stablecomplexes, so that the tightest binding combinations are enriched. Ifthe bead coferon contains a Veracode™ and the solution coferon a DNAtag, both ligands may be readily identified. Veracode™ beads which arefluorescently labeled may be sequestered into individual wells usingFACS sorters or 454 sequencing instruments. DNA tags bound by individualbeads may then be individually amplified and identified. If the solutioncoferon does not contain an identification tag, the ligand can still beidentified by using the same set of diversity elements on the beads andin solution. Only the winning pairs will provide the strongestfluorescent signal, and, if it is just a few beads, the combinations maybe tested individually. The coferon pairs selected by this protocolshould have affinities to the target in the nanomolar range.

A second version of a screen, as shown in FIG. 21, is very similar tothe first version, using diversity elements on both beads and insolution. Fluorescently labeled target protein is added to the beads andin solution coferons, and after a suitable period of panning, wellscontaining fluorescently labeled beads are identified. If the beads arein a single chamber, the Veracode™ is identified for the fluorescentlylabeled beads. In both cases, the in solution coferons are identified byPCR amplification and sequencing of the DNA tags. The PCR amplified tagsmay also be used to re-synthesize the diversity elements, such thatevolutionary principles are used to select the winning pairs ofcoferons. Alternatively, each of the top ligands may be re-synthesizedand can now be attached through a series of connectors that vary insize, flexibility, or for circular diversity elements, the size of themacrocycle. A further variation on this theme would be to regenerate notonly the original diversity elements, but minor variations (for examplevary just one amino acid residue at a time from a cognate sequence) aswell, to be combined with the diverse set of connector elements. Thisrefined set of coferons would be re-screened in the presence of thefluorescently labeled target protein at the appropriate concentrationsto allow for selection of the tightest binding combinations. The sameprinciples of dynamic combinatorial chemistry described above wouldapply. The winning pair of coferons are identified by their Veracode™and DNA sequence tags. The coferon pairs selected by this protocolshould have affinities to the target in the nanomolar range.

A third version of a screen, as shown in FIG. 22, is similar to thefirst version, using diversity elements on two sets of coferons insolution. Here a first set of DNA encoded coferon is either passed overor cycled through a column containing the target protein. After bindingthe library and an optional step of washing with buffer to removenon-specific binders, the diversity elements are identified by PCRamplification and sequencing of the DNA tags. This approach identifiesthe diversity elements that bind most tightly to the target (withaffinities in the micromolar to nanomolar range). The PCR amplicons areused to resynthesize the top diversity elements. (In a variation, theabove column selection procedure may be repeated to further enrich forthe tightest binding diversity elements.) This first set of selecteddiversity elements is now combined in solution with a second completeset of diversity elements whose coferons bind to the first set ofcoferons. These coferon pairs are then panned on beads containing theprotein target. The principles of dynamic combinatorial chemistry wouldselect for and amplify those coferon pairs that bind most tightly to thetargets on the beads. After a suitable period of time, the unboundcoferons are removed, with the optional step of washing the beads tofurther remove non-specific binding coferons. The panning may be done inwells that have filter bottoms, or, alternatively, the beads containingthe target proteins may be magnetic for magnetic capture to facilitatewashing steps. The diversity elements for each set of coferons areidentified by PCR amplification and sequencing of the DNA tags.Alternatively, each of the top ligands identified in the first screenmay be re-synthesized and can now be attached through a series ofconnectors that vary in size, flexibility, or for circular diversityelements, the size of the macrocycle. A further variation on this themewould be to regenerate not only the original diversity elements, butminor variations (e.g., vary just one amino acid residue at a time froma cognate sequence) as well, to be combined with the diverse set ofconnector elements. This refined set of coferons would be re screened inthe presence of the complete second coferon library in solution to findbinders to the target protein on beads. The same principles of dynamiccombinatorial chemistry described above would apply. The winning sets ofcoferon pairs are identified by their DNA sequence tags. A different setof universal primers may be used to amplify and distinguish the DNA tagsfrom the first coferon diversity set compared to the second coferondiversity set. If multiple sets of coferons from different clades areselected, the individual combinations can be determined byresynthesizing individual coferons, and trying each set in independentbinding assays. As above, the second set of coferons may also beoptimized by introducing variations in the connectors and diversityelements. Thus, evolutionary selection pressures may be applied on bothsets of the coferon diversity elements. The coferon pairs selected bythis protocol should have affinities to the target in the nanomolar topicomolar range.

A fourth version of the screen, as shown in FIG. 23, is a variation ofthe first and third versions. In this case, the target is a protein witha known binding pocket, for example a tyrosine kinase. Here, moleculesknown to or inferred to fit within the binding pocket (and chemicalvariants whose structure would occupy a similar 3-dimensional space) areattached to a series of connectors that vary in size and flexibility. Itis assumed (but should also be experimentally verified) that themajority of members of this library would bind to the target withmicromolar or even nanomolar affinities. This is the equivalent of thefirst round of screening in the above first and third versions (FIGS. 20and 22). Subsequently, this first library of binding pocket ligands iscombined with the second library of coferons with various diversityelements. The pairs of binding elements are screened as described above.The coferon pairs selected by this protocol should inhibit enzymaticfunction if the binding pocket also has an active site, and haveaffinities to the target in the nanomolar to picomolar range.

A fifth version of the screen, as shown in FIG. 24, combines elements ofthe above versions. In the first version (FIG. 20), monomer diversityelements on beads are screened for binding to fluorescently labeledtarget, while in the third version (FIG. 22), monomer DNA taggeddiversity elements are screened for binding to immobilized target onbeads or a solid support. In both cases, the initial selection is for amonomer binding ligand to the target. Under these conditions, theconnector and linker element would be anticipated to play a minor rolein the binding affinity. These initial top ligands are then redeployedwith a series of different connectors and now selected again in thepresence of a second library of diversity elements to achieve evenbetter binding. These screens are based on the principle that initialstronger binding monomer diversity elements would most likely beselected for in the strongest coferon dimer pair, and that a step-wiseselection for the tightest binding elements would be successful. In thefifth version of the screen (FIG. 24), two sets of coferon librarieswith encoded DNA barcodes are combined in solution to form dimer pairs.The DNA barcodes have the capacity for additional levels of diversitythat were used in synthesis of the ligands. The coferon libraries arescreened directly for binding to target immobilized on beads, and thetightest binding ligand pairs are enriched due to dynamic combinatorialchemistry. The winning ligand pairs are identified via PCR amplificationof the encoded barcodes, and these amplicons are then used for anadditional round of ligand synthesis. However, in this next round,additional diversity is added with a series of different connectors,which are encoded by using regions of the DNA barcode that were not usedfor generating the original ligand diversity. The products of this roundof synthesis have been enriched for the tightest ligand pairs selectedin the initial panning step, yet have additional diversity added for thesecond panning step. This type of chemical screening-selection mimicsthe type of evolutionary selection that occurs on eukaryotic organismsthat reproduce by sexual means and thus are constantly reassortingchromosomal pairs. This two-step screening-enrichment allows for agreater degree of diversity elements at varying distances andorientation with respect to each other to be interrogated in the overallselection process. The coferon pairs selected by this protocol shouldhave affinities to the target in the nanomolar to picomolar range.

The recent work of Whitesides (Krishnamurthy, et al., J. Am. Chem. Soc.129:1312-1320 (2007), which is hereby incorporated by reference in itsentirety) and Neri laboratories (Melkko, et al., Nat. Biotechnol.22(5):568-574 (2004), which is hereby incorporated by reference in itsentirety) suggest that diversity elements will bind to a target withalmost as high binding affinity when attached through a flexibleethylene glycol linker as when attached by a rigid linker of theprecisely correct geometry. This finding allows one to liberate theprocess of screening for the best diversity elements of a given targetfrom the exact linker element (and/or connector) design used in thefinal coferon drug. Thus, diversity elements may be optimized for agiven target using a set of linker elements which have a favorableequilibrium between the monomer and dimer state; i.e. one that favorsthe dynamic combinatorial chemistry selection process. Subsequently,these same or different linker elements may be optimized using eitherflexible or more rigid connectors between the diversity elements(ligands) and the linker elements to optimally bind the target.

For example, when performing in vitro screening of diversity elementsbinding to a target protein, it would be advantageous to use a firstlinker element containing an aldehyde or ketone, and a second linkerelement containing a primary or secondary amine. These two linkerelements readily form the highly reversible Schiff base in the absenceof target at the concentrations of diversity elements used forscreening. There is a high concentration of primary amines free insolution (lysine) and in proteins. Thus, when using a coferon monomercontaining a primary amine, it is important for the companion aldehydeor ketone containing coferon monomer to find its partner on the surfaceof the target molecule. As an added note of caution, theamine-containing linker element may react with sugars when in thealdehyde or ketone tautomeric form. However, these linker elements wouldnot be preferred in the final coferon designs, since there is a highconcentration of primary amines free in solution (lysine) and inproteins, and thus it may be difficult for the first aldehyde or ketonecontaining coferon to find its partner containing a primary amine insolution. Further, the primary amine in the second linker element mayalso react with aldehydes and ketones present in sugars that are in thelinear isomeric form. However, if the primary amine is two carbons awayfrom a thiol group (which may be in the protected disulfide form outsidethe cell), then it has the potential to form an essentially irreversiblethiazolidine linker in the final coferon dimer. The thiazolidine linkeris an excellent example of a linker element that may be activated uponentering a cancer cell and then form an essentially irreversible bondwith its partner coferon.

In-silico screening can be performed with the aim of limiting the numberof different diversity elements tested when performing in vitroscreening. In-silico screening would be performed with either a knowndiversity library, or with an in-silico library, where the potentialstructures are all known or may be calculated. Such molecules can bevirtually screened by matching the 3D models with the target proteinstructure. In-silico screening would allow the testing of huge virtuallibraries of different diversity elements on different scaffolds, withthe aim of eliminating the vast majority of potential diversitystructures and focusing on a reasonable number of promising leads. Sincethe process of building such combinatorial libraries in vitro isstraightforward, an in-silico pre-screen has the potential to acceleratethe process of identifying lead candidate coferons. This will beespecially useful for screening diversity elements in multimericcoferons.

Identification of a first diversity element may assist in identifying asecond diversity element that binds the target adjacent to the firstdiversity element. Likewise, use of a known ligand as the firstdiversity element will assist in identifying a second diversity elementthat binds the target adjacent to the first diversity element. Thisapproach may improve an existing drug by taking advantage of the largersurface area that a coferon pair can use to bind onto the target, thusimbuing the coferon with higher affinity or better specificity, or both.

The coferon concept takes advantage of having three weak interactionstake place simultaneously as follows: (i) coferon 1 to coferon 2; (ii)coferon 1 to protein; and (iii) coferon 2 to protein, which results in avery strong interaction between the protein and the two coferonpartners. The coferon interaction may be strengthened by covalent bondsbetween the coferons. The reactive groups on the coferons are chosensuch that they are mostly unreactive with cellular molecules or offtarget proteins. If they do react with cellular components, suchreactions should be reversible and non-toxic.

Just as the interactions between the coferons may be strengthened bycovalent bonds, so too, the interactions between the coferons and theprotein partners may also be strengthened by incorporating reactivegroups within the diversity elements that bind the protein target. Forexample, a ketone or aldehyde in the correct orientation may form aSchiff base with a lysine on the protein target. Another example wouldbe reaction of a coferon boronic acid group with a threonine or serineresidue on the protein target or carbohydrate hydroxyl groups onglycoprotein targets. Coferons containing boronic esters could link witheach other as well as with multiple sites on the carbohydrate portion ofglycoproteins. Either one or both of these events would significantlyshift the equilibrium towards coferon dimer binding to its target. Suchdesigns are dependent on judiciously placed amino acid residues on thetarget protein. Although there is a risk of non-specific reactionbetween a reactive group on the coferon drug and an incorrect target,since the rest of the diversity element would not provide any additionalbinding energy, such an off-target effect would be quickly reversible.

The above principle extends even further when applied to coferonmultimers, and especially to coferon multimers that bind multimericprotein targets. Multiple weak interactions add to the binding affinityof the overall coferon complex to the correct target.

When screening for the best coferons, either one of the coferons or theprotein target is on a solid support (bead), with coferons binding toeach other and/or the protein target. The bound coferons are inequilibrium with the coferons in solution, both binding and coming apartthrough their linker element moieties. Meanwhile, the protein targetsare binding and dissociating with coferons in solution and on the solidsupports. The most stable complexes of bead coferon to solution coferonto target protein are removed from this equilibrium. The concentrationof these components in solution has now decreased, so they dissociatefrom less stable complexes. This now drives the equilibrium towardsforming even more of the most stable complexes, so that the tightestbinding combinations are enriched.

For this screening process to work most effectively, the coferonmonomers need to efficiently cycle between the monomeric and dimeric (ormultimeric) state. This will allow for the greatest number ofcombinations to be tested, and also for enriching the best bindingcombinations onto the solid support.

However, as mentioned above, some linker elements may associate slowlyuntil brought in close proximity by the target, but once they associateand form one or more covalent (i.e. hemiacteal) or ionic bond (i.e.through two coferons chelating the same zinc ion), they do notdissociate easily. Thus these types of reactions are essentiallyirreversible. While such a property of a coferon may be desirable forlinker elements in the final drug molecule, they would inhibit thescreening process.

In order to use such linker elements during the dynamic combinatorialchemistry screening process, it is preferable for the dissociationprocess to occur as rapidly as the association process. One approach isto change the assay conditions, for example, low pH will favordissociation of hemi-acetals. Another approach is to use linker elementswith the same geometry, but now unable to form all the potentialcovalent bonds.

A new approach is to cycle between conditions that favor formation ofdimers and multimers, and conditions that favor dissociation tomonomers. Herein, this approach is termed cyclic combinatorialchemistry, or C3 screening.

Consider a coferon pair that associates quickly at pH 9, and dissociatesquickly at pH 5. The coferon association is initiated by combining abead-library and a solution library of coferons with the protein target,for example in a phosphate buffer at pH 9. As library members cometogether, some pairs will favor binding to the protein target. Othernon-productive pairs will also come together. The pH may now be titrateddown to pH of 5 by addition of acid. Under these conditions, coferonsthat are not bound to the target will dissociate, but coferons bound tothe target are held in place, and do not dissociate. Subsequently the pHis shifted back to pH 9. Now fresh combinations of coferon pairs form,and again, the pairs that favor binding of the protein will accumulatemore protein on the beads or particle. This process may be repeateduntil sufficient (fluorescently) labeled protein accumulates on thebeads containing the best coferon pairs. One caveat with this approachis that the ion concentration in the solution keeps increasing (forexample, if HCl and NaOH are used to decrease and increase the pH,respectively, then NaCl will accumulate with each cycle). On thepositive side, higher salt concentrations will select for more specificbinding. Further, this process is easy and amenable to automation.

As another example, consider coferons that pair through a Zn²⁺ cofactor.Addition of 1 mM ZnCl₂ will allow the coferons to dimerize, with themore favorable pairs binding to the target. Addition of a suitable zincchelating agent (such as 1 mM EDTA) will be able to displace coferonsfrom the zinc so the coferons dissociate into monomers. The chelatingagent should not be strong enough to dissociate the zinc when the twocoferons are held in place by binding a target. Alternating addition of1 mM ZnCl₂ and 1 mM EDTA will cycle the “free” Zn²⁺ cofactor in solutionbetween approximately 1 mM and 0 mM, cycling the coferons between thedimer (or multimer) and the monomer states. As noted previously with pHcycling, this will eventually accumulate Zn-EDTA (in the process,forming NaCl if the original EDTA was in the disodium salt). Thisprocess is also amenable to automation.

To avoid accumulating salt, alternative approaches may be used tomodulate pH or divalent metal concentration. For example, the chelatingmoiety may be attached to a solid support and brought in contact withthe coferon screening solution by circulating the screening solutionpast the solid support. Coferon on beads or particles may be separatedfrom chelator beads or particles by using different size beads orparticles, or using paramagnetic beads or particles. To modulate pH,organic molecules that act as buffers may be attached to a solidsupport. Among these are “Good's buffers”, which can stabilize pH valuesover very precise ranges. The coferon screening solution may becirculated between two chambers, each containing the solid support withthe organic molecule that will buffer the screening solution to theright pH. In both of these examples, the solid support may eventuallybecome saturated (with divalent cation, or exceed it's bufferingcapacity), and thus may need to be replaced after a certain number ofcycles. As before, this process is also amenable to automation.

In the above examples, the binding of coferons to each other iscontrolled by the concentration of a positively charged ion or cation:H⁺ or Zn²⁺. Certain membranes are permeable to small molecules and ions.The Nafion-117 membrane is permeable to H⁺, and cations such as Li⁺,Mg²⁺, Zn²⁺, Na⁺, and K⁺; but impermeable to coferons, anions, buffers,large cations, nucleic acids, peptides and proteins. This membrane maybe used in a device that allows for cyclic combinatorial chemistry.

In one embodiment (See FIGS. 25 and 26), the membrane separates an uppercompartment A from a lower compartment B. Compartment A contains beads,coferons, buffer (such as PIPS, TEEN, or PIPPS), and target protein. Thebuffer is chosen to provide the desired pH range based on pKa values(PIPPS buffer has a pKa1 3.85; pKa2 7.99; PIPES buffer has a pKa1 2.7;pKa2 6.81; and TEEN buffer has a pKa1 6.69; pKa2 10.10). At the higherpH, the coferons are more stable in the multimer form, while at thelower pH, the coferons dissociate to form monomers—unless they are boundto the protein target, where they remain as multimers.

Compartment B is used to wash in and out different buffers in reservoirsC-E. Reservoir C contains an aqueous wash solution. Reservoir D containsH⁺ or a low pH buffer. Reservoir E contains NaOH (or equivalent base),or a high pH buffer. During cycling, ionic strength and amount of bufferremain unchanged in Compartment A. Cation and water exchange across theNafion-117 membrane between compartments A and B is mediated by pistonpumps, stirring liquid in either compartments, applying pressure, orcombinations thereof. Cations cycle between H+ and Na+ (or equivalentcation).

If the coferons bind through a Zn²⁺ cofactor, then reservoir D containsthe Zn²⁺ and reservoir E contains a chelator, such as EDTA. Duringcycling, ionic strength and amount of buffer remain unchanged inCompartment A. The Zn²⁺ and Na⁺ cations (and water) exchange across theNafion-117 membrane between compartments A and B is mediated by pistonpumps, stirring liquid in either compartments, applying pressure, orcombinations thereof. Cations cycle between Zn²⁺ and Na⁺.

The above design is amenable to a multiple well format and automation. A24 well microtiter plate may be constructed from 2 parts: The top parthas cylindrical openings in 24 well format. The bottom part has shallowwells and grooves from a single entry port on the front splitting into24 lines going into each well, and 24 lines (grooves) out of each wellcoming together at a single exit port in the back. Such a design can bemanufactured very quickly in a simple stamping process. The top andbottom part are welded together with the Nafion-117 membrane in betweenthem. The entry and exit ports both have valves and are attached topiston pumps.

Since the 24 top wells are open, they can be filled with coferons,beads, fluorescent target protein, etc. using a multi-channel pipette ora robotic platform.

The bottom of the wells can be filled with the appropriate reagents byopening the entry and exit valves, and moving the two piston pumps inthe same direction. The simplest way to accelerate the exchange is tohave the entire device on a rotating platform (microtiter plate shaker).Alternatively, magnetic agitation (stirring) may be used. If it isnecessary to speed up the process, the exit pump can be closed, and thevolume of all 24 top wells will increase when the entry pump keepspumping. To decrease the volume of the 24 top wells, the entry valve isclosed, and the exit valve is opened and the pump withdraws fluid. Thisdesign also makes it easy to transfer a number of reactions into asecond microtiter plate for bulk washing away unbound coferons etc.

A fluorescent chelator or dye may be used to monitor the zincconcentration or pH. Examples of fluorescent zinc chelator and somefluorescent pH dyes are: TFLZn,4-(6-Methoxy-8-quinaldinyl-aminosulfonyl)benzoic acid potassium salt;HPTS, 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt;umbelliferone-3-carboxylic acid, 7-hydroxycoumarin-3-carboxylic acid;and 5(6)-carboxynaphthofluorescein.

After the selection is complete, the dye or fluorescent group may bewashed away so that it does not interfere with scoring of the beads forthose that bound labeled target protein.

The dyes can also be linked to a solid support to make it easy to readand separate from coferon beads (although a separation step may not beneeded).

It may be useful to verify the rate and efficiency of exchange using amodel system. One such model system would use iminobiotin as the ligand,and fluorescently labeled streptavidin as the target protein. Afunctional coferon would be synthesized containing the linker elementconnected to the iminobiotin via a flexible linker, i.e. ethylene glycolchain. When synthesizing this functional coferon on a solid support,spacing would be sufficiently distant to minimize two coferons in closeenough proximity to bind to the same streptavidin target. Anon-functional coferon would be synthesized containing the linkerelement connected to another unrelated small molecule or just an aminegroup via an ethylene glycol chain. The functional coferon containingbead would be mixed in with a 1,000-fold excess of beads containingnon-functional coferon. Likewise, the functional coferon in solutionwould be mixed in with a 1,000-fold excess of non-functional coferon insolution. In the example here, the solution coferon can only make dimersor multimers with the bead-bound coferon.

In the presence of fluorescently labeled streptavidin, two functionalcoferons, one on the bead, the other in solution bind to the target andprovide a small amount of fluorescent label to the single bead. Withrepeated cycling (100 to 1,000 cycles), the amount of fluorescent signalon the functional coferon bead should steadily increase. Comparingdifferent cycling conditions will help determine the optimal cyclingtimes and pH or cation concentrations.

Considerations for Screening Coferons Binding to Targets

In FIGS. 2.1 through 2.16, coferons are described from monomers tohexamers; design components and variations, suitable for synthesis,screening, and therapeutic use.

In consideration of the screening process, the following encryptionformats—illustrated below using the simplest case of forming dimersbetween “A” and “B” coferons—may be considered:

Multiple A Coferons with Multiple B Coferons.

1. Multiple A coferons and multiple B coferons both contain DNAencryption. DNA encryption of coferons, using either DNA targetedsynthesis or DNA directed synthesis has been described in theliterature. The coferons are blended with an immobilized target, andafter the appropriate incubation period are washed off. Addition of theappropriate primers allows for PCR amplification of the DNA bar-codes,and sequence analysis allows for identification of the ligands in boththe “A” and “B” coferons.

2. Multiple A coferons and multiple B coferons, A coferons are on beads,B coferons in solution. In this variation, the A coferons are on encodedbeads, and the B coferons have a DNA encryption or no encryption.Fluorescently labeled target is added, and beads that becomefluorescently labeled in the presence of B coferons are isolated. Ligandon the bead may be identified through (i) optical bar-coding of thebead, (ii) mass-tag bar-coding of the bead, (iii) DNA bar-coding of thebead. B coferon may be identified using the DNA bar-code (as describedabove) or by mass spectroscopy. Alternatively, the selection is repeatedwith the B coferons are on encoded beads, and the A coferons in solutionto find the matched pairs.

Single A Coferon with Multiple B Coferons.

1. Single A coferon with multiple B coferons, B coferons on beads. Theseare conditions where the A coferons are either known ligands, or havebeen pre-screened against the target so there are a limited number of Acoferons to test, compared with a vast excess of B coferons.Fluorescently labeled target is added, and beads that becomefluorescently labeled in the presence of B coferons are isolated. Ligandon the bead may be identified through (i) optical bar-coding of thebead, (ii) mass-tag bar-coding of the bead, (iii) DNA bar-coding of thebead.

2. Single A coferon with multiple B coferons, both coferons in solution.These are conditions where the A coferons are either known ligands, orhave been pre-screened against the target so there are a limited numberof A coferons to test, compared with a vast excess of B coferons. Forthe purposes of this example, the coferons are used in a whole-cellassay, where inhibition of the target is lethal, and thus amenable toHTS. Consider 96 A coferons to be tested in combination with 9,600 Bcoferons (One 96 well plate vs. 100×96 well plates). Ordinarily, thatwould require 921,600 assays (=2,400×384 well plates). However, bypooling coferons by rows and columns, the total number of screens forthe A coferon plate vs 1 B coferon plate would be 8 A rows×12 Bcolumns+12 A columns×8 B rows=192 wells. If one coferon combinationworked, then one of the first 96 well assays will give a positiveresult, and one in the second 96 well assays will also give a positiveresult, allowing identification of the correct row and column in boththe A and B plate to identify the precise coferon pair that works. Ifthis process is now repeated for each of the 100 B plates, this wouldrequire 19,200 assays (=50×384 well plates). Since dynamic combinatorialchemistry selects the tightest binding inhibitors, the pooling strategyallows for almost a 50-fold reduction in the number of HTS assaysrequired.

Single A Coferon with Single B Coferon.

1. Single A coferon with single B coferon, with coferon biologicalactivity determined using whole-cell assays. Examples of biologicalreadout are provided below. In these schemes, both coferons are insolution. The identity of the coferon is given by the location of thewell where the ligand was synthesized, for example by split synthesisprotocols, without re-pooling. Such assays may be compatible with thepooling strategies described above. Alternatively, where assays are notcompatible with pooling, ultra high-throughput assays may be developedusing nano-droplet (Raindance) technology. Such technology can generate3,000 droplets per second. Consider the example above of 96 A coferonsto be tested in combination with 9,600 B coferons, where the whole-cellassay generates a fluorescent signal. The A coferons are in 1×96 wellplate, each well containing a 100,000 beads with a unique barcode andthe A coferon attached to the bead. The B coferons are in 25×384 wellplates, each well containing a 1,000 beads with a unique barcode and theB coferon attached to the bead. In practice, either the A or B coferonplate may pool the coferons by using split synthesis protocols, withre-pooling, provided the barcodes are attached to the beads. All the Acoferons are pooled together and emulsified in oil such that each beadis in its own nanodrop. Likewise, all the B coferons are pooled togetherand emulsified in oil such that each bead is in its own nanodrop. The Acoferon droplets and B coferon droplets are fused, each fused dropletcontaining one bead each for a total of 9,600,000 droplets. This process(not including setup) takes 3,200 seconds, or just under an hour. Thesedroplets are then exposed to light (or heat, or reagent that may besubsequently neutralized if needed to be biologically compatible) torelease the coferons from the beads. Subsequently, the droplets arefused with new droplets containing the cells with the biological targetwhose inhibition/activation will result in a change in fluorescentsignal. This second droplet fusion will also take just under an hour,and this may be followed by a period of incubation to allow the coferonsto enter the cells and bind the intended target, resulting in thebiological readout. The droplets are placed in a flow sorter, such thatthe fluorescently altered droplets are separated. Dilution into 384 or1536 well plates, such that a given well has one or less nanodropletscontaining the original bead pair, followed by addition of aqueoussolution and centrifugation separates the aqueous layer from thehydrophobic oil and allows for PCR amplification and sequence analysisto identify the winning coferon ligands. If the bar-codes are mass tagsattached to the beads, they may be identified by mass spectroscopy.

2. Single A coferon with single B coferon, with coferon bindingdetermined using in vitro readout. Examples of in vitro readout areprovided below. In these schemes, both coferons are in solution. Theidentity of the coferon is given by the location of the well where theligand was synthesized, for example by split synthesis protocols,without re-pooling.

Coferon Binding Determined Using in vitro Readout.

Two screens, termed “AlphaScreen” and “AlphaLISA” have been developed(sold by Perkin-Elmer) to measure cell signaling, includingprotein:protein, protein:peptide, protein:small molecule orpeptide:peptide interactions. The assays are based on detecting theclose proximity of donor beads containing a first molecule or proteinthat binds to a second molecule or protein on the acceptor beads.Singlet oxygen molecules, generated by high energy irradiation of donorbeads, travel over a constrained distance (approx. 200 nm) to acceptorbeads. This results in excitation of a cascading series of chemicalreactions, ultimately generating a chemiluminescent signal. (Eglen, et.al., Curr. Chem. Genomics 1:1-19 (2008), which is hereby incorporated byreference in its entirety).

The donor bead contains phthalocyanine. Excitation of the donor bead bya laser beam at a wavelength of 680 nM allows ambient oxygen to beconverted to singlet oxygen. This is a highly amplified reaction sinceapprox. 60,000 singlet oxygen molecules can be generated and travel atleast 200 nm in aqueous solution before decay. Consequently, if thedonor and acceptor beads are brought within that proximity as aconsequence of protein:protein, protein:peptide, or protein:smallmolecule interactions, energy transfer occurs. Singlet oxygen moleculesreact with chemicals in the acceptor beads to produce a luminescentresponse. If the acceptor bead contains Europium, as in the AlphaLISAassay, an intense luminescence is emitted at a wavelength of 615 nm.(Eglen, et. al., Curr. Chem. Genomics 1:1-19 (2008), which is herebyincorporated by reference in its entirety).

For the purposes of the discussion below, this system will be referredto as linking various proteins, fragments or molecules on donor andacceptor beads. Such linking may be chemical in nature, or may be due totight binding of a tethered ligand, such as if the donor bead is coatedwith strepavidin and the donor molecule or protein has a biotin attachedto it. There are many systems for binding recombinant proteins to beads,including His-Tag, Myc-Tag, GST fusions, Maltose binding protein (MBP)fusions.

A. Identifying Initial Sets of Coferon A Ligands that (Weakly) Bind tothe Target Protein

Target protein is linked or bound to the donor bead. A generic coferonB, containing a linker element that binds the linker element of coferonA is attached to the acceptor bead. A generic ligand may contain thescaffold and then the simplest diversity element in all the diversitypositions, for example, alanine if the diversity positions are filledwith amino acid moieties. An HTS assay is setup containing acceptor anddonor beads in each well, with from 1 to 100 or even 1,000 or morecoferon A variants added to each well. The number of variants willdepend on the background level and hit level, determined experimentally.Likewise, the number of “generic” variants that can be tested within thesame well may range from 1 to 100 or more. Since dynamic combinatorialchemistry takes place, the acceptor bead will bind those variants thatbind the donor bead the tightest, as more than one protein will interactwith more than one coferon pair to form more than one bridge to theacceptor bead. By using different sets of pools (i.e. rows vs. columns)a large number of potential binders may be rapidly tested.

B. Identifying Optimized Coferon B Ligands that Ppair with the InitialSets of Coferon A Ligands to Tightly Bind to the Target Protein

Target protein is linked or bound to the donor bead. The initials setsof coferon A ligands, (containing a linker element that binds the linkerelement of the test coferon B ligands) are attached to the acceptorbeads. An HTS assay is set up containing acceptor and donor beads ineach well, with from 1 to 100 or even 1,000 or more coferon B variantsadded to each well. The number of variants will depend on the backgroundlevel and hit level, determined experimentally. The strongest bindingcoferon B ligands will give the brightest signals. As above, whentesting more than one coferon B ligand per well, use of different setsof pools (i.e. rows vs. columns) allow a large number of potentialbinders to be rapidly tested.

C. Identifying Coferon Dimers that Enhance Binding of Two Proteins withWeak or no Binding Affinity to Each Other

Target protein 1 is linked or bound to the donor bead. Target protein 2is linked or bound to the acceptor bead. To identify a new weak bindingpartner to a given target protein, a yeast two-hybrid or other fish-baitprotein complementation assay is set up, with both weak and strong hitsidentified. An HTS assay is set up containing acceptor and donor beadsin each well, with from 1 to 10 or even 100 or more coferon A & B dimervariants added to each well. The number of variants will depend on thebackground level and hit level, determined experimentally. The coferondimers that best enhance binding of the two proteins to each other willgive the brightest signals. If necessary, candidate coferon A and Bmonomers that bind either or both protein targets may be identified asin procedure A.

D. Identifying Coferon Dimers that Further Enhance Binding of TwoProteins with Medium to Strong Binding Affinity to Each Other

Target protein 1 or a mutant variant with weaker binding is linked orbound to the donor bead. Target protein 2 or a mutant variant withweaker binding is linked or bound to the acceptor bead. If the originalproteins are used, they are linked to the beads at low concentration.Often some structural or sequence information is available to guidealanine scanning or targeted mutagenesis to generate variants with thepotential to bind weakly. To identify mutations that convert a strongbinding partner into a weak binding partner to a given target protein, ayeast two-hybrid or other fish-bait protein complementation assay is setup to test mutant variants, with both weak and strong hits identified.An HTS assay is set up containing acceptor and donor beads in each well,with from 1 to 10 or even 100 or more coferon A & B dimer variants addedto each well. The number of variants will depend on the background leveland hit level, determined experimentally. The coferon dimers that bestenhance binding of the two proteins to each other will give thebrightest signals. The winning coferon dimer sets are then retested todetermine which set enhances binding of the wild-type proteins to eachother.

E. Identifying Coferon Dimers that Inhibit Binding of Two Proteins toEach Other

Target protein 1 is linked or bound to the donor bead. Target protein 2is linked or bound to the acceptor bead. An HTS assay is set upcontaining acceptor and donor beads in each well, with from 1 to 10 ormore coferon A & B dimer variants added to each well. The number ofvariants will depend on the background level and hit level, determinedexperimentally. The coferon dimers that best inhibit binding of the twoproteins to each other will give the weakest signals. If necessary,candidate coferon A and B monomers that bind either protein targets inthe absence of the other protein may be identified as in procedure A.

F. Identifying Coferon Dimers that Inhibit Binding of Two Proteins toEach Other

Target protein 1 is linked or bound to the donor bead. Target protein 2is either added in solution, or linked or bound to neutral beads. A weakor medium binding partner of target protein 1, or an antibody that bindsto target protein 1 is linked or bound to the acceptor bead. An HTSassay is set up containing acceptor and donor beads, as well assufficient target protein 2 in each well, such that target protein 2interferes with binding of the proteins on the acceptor and donor beadsresulting in low or background level signal. Addition of from 1 to 10 ormore coferon A & B dimer variants that bind to target protein 2 in sucha way as to disrupt binding to target protein 1, allowing for binding ofthe protein on the acceptor bead to the donor bead, and thus generatingpositive signal. The number of variants will depend on the backgroundlevel and hit level, determined experimentally. The coferon dimers thatbest inhibit binding of the two proteins to each other will give thestrongest signals. If necessary, candidate coferon A and B monomers thatbind target protein 2 in the absence of the other protein may beidentified as in procedure A.

G. Identifying Coferon Dimers that Inhibit Binding of Two Proteins toEach Other

The inverse of the above procedure may be performed using target protein2 linked or bound to the donor bead, and target protein 1 either addedin solution, or linked or bound to neutral beads. In this procedure, aweak or medium binding partner of target protein 2, or an antibody thatbinds to target protein 2 is linked or bound to the acceptor bead.Again, if necessary, candidate coferon A and B monomers that bind targetprotein 1 in the absence of the other protein may be identified as inprocedure A.

H. Identifying Coferon Dimers that Inhibit Binding of Two Proteins toEach Other, Using a Helper Protein

Target protein 1 is linked or bound to the donor bead. Target protein 2is linked or bound to the acceptor bead. A helper protein may have weakor no affinity to target protein 1. An HTS assay is set up containinghelper protein, acceptor and donor beads in each well, with from 1 to 10or more coferon A & B dimer variants added to each well. The number ofvariants will depend on the background level and hit level, determinedexperimentally. The coferon dimer that enhances binding of the helperprotein to target protein 1, and thus best inhibits binding of the twotarget proteins to each other will give the weakest signals. Ifnecessary, candidate coferon A and B monomers that enhance binding ofthe helper protein to target protein 1 in the absence of the otherprotein may be identified as in procedure C.

I. Identifying Coferon Dimers that Inhibit Binding of Two Proteins toEach Other, Using a Helper Protein

Target protein 1 is linked or bound to the donor bead. Target protein 2is either added in solution, or linked or bound to neutral beads. A weakor medium binding partner of target protein 1, or an antibody that bindsto Target protein 1 is linked or bound to the acceptor bead. A helperprotein may have weak or no affinity to Target protein 2. An HTS assayis set up containing acceptor and donor beads, as well as sufficienttarget protein 2 and helper protein in each well, such that targetprotein 2 interferes with binding of the proteins on the acceptor anddonor beads resulting in low or background level signal. Addition offrom 1 to 10 or more coferon A & B dimer variants that enhance bindingof the helper protein to target protein 2 in such a way as to disruptbinding to target protein 1, allowing for binding of the protein on theacceptor bead to the donor bead, and thus generating positive signal.The number of variants will depend on the background level and hitlevel, determined experimentally. The coferon dimer that enhancesbinding of the helper protein to target protein 2, and thus best inhibitbinding of the two target proteins to each other will give the strongestsignals. If necessary, candidate coferon A and B monomers that enhancebinding of the helper protein to target protein 2 in the absence of theother protein may be identified as in procedure C.

J. Identifying Coferon Dimers that Inhibit Binding of Two Proteins toEach Other, Using a Helper Protein

The inverse of the above procedure may be performed using target protein2 linked or bound to the donor bead, and target protein 1 either addedin solution, or linked or bound to neutral beads. In this procedure, aweak or medium binding partner of target protein 2, or an antibody thatbinds to target protein 2 is linked or bound to the acceptor bead. Ahelper protein may have weak or no affinity to target protein 1. Again,if necessary, candidate coferon A and B monomers that enhance binding ofthe helper protein to target protein 1 in the absence of the otherprotein may be identified as in procedure C.

Coferon Biological Activity Determined Using Whole-Cell Assays.

The last few years has seen an explosion of biological assays designedto study protein signaling and protein-protein interactions in wholecells. Many of these are based on protein complementation assays (PCA's)that reconstitute activity of two peptide chains to form a functionalreporter protein, which generates either a fluorescent orchemiluminescent signal. Proteins have evolved to code for all theinformation needed to fold into stable 3-dimensional structures. In somecases, the complementary N-terminal and C-terminal peptide chains canfold independently, and find each other to form a functional (reporter)protein. However, kinetically this process competes with non-specificaggregation, so in many cases expression of complementary N-terminal andC-terminal peptide chains in a cell does not lead to reconstruction ofactivity. PCA works by fusing interacting proteins to the fragments,which increase the effective concentration of the two fragments, thusfavoring the correct folding over any non-productive process. Additionof coferon drugs that would interfere with the two proteins frominteracting with each other would lower the effective concentration ofthe two fragments with each other, and thus cause a disruption or lossof signal from the complementing reporter protein fragments.

One of the oldest forms of protein complementation in based on thealpha-peptide complementation of the enzyme beta-galactosidase.DiscoveRx has developed this enzyme fragment complementation (EFC)technology into a cell-based luminsescent platform. Beta-galactosidaseis active as a tetramer, but when missing the N-terminal 60 amino acidpeptide forms only dimers, which are inactive. By reintroducing thealpha-peptide into the protein, it forms the tetramer and revivesactivity. Two forms of the alpha-peptide are commercially available,ProLabel™ (DiscoverRx Corp., Fremont, Calif.) with higher affinity tothe C-terminal enzyme acceptor protein, and ProLink™ (DiscoverRx Corp.,Fremont, Calif.), with lower affinity, and thus optimized to detectprotein-protein interactions. By engineering G-Protein

Coupled Receptors (GPCRs) to contain the ProLink peptide on one of theirtermini, and using an engineered beta-arrestin to contain the C-terminalenzyme acceptor protein, DiscoveRx has developed an assay fordrug-activation of GPCR with EFC readout in the form of achemiluminsescent signal. Similarly, the ProLabel tag has been used tomeasure protein expression, degradation, secretion and translocation fora variety of drug discovery target classes.

An alternative approach is marketed by Invitrogen (Carlsbad, Calif.) andtermed “GeneBLAzer Technology”. GeneBLAzer Technology uses amammalian-optimized beta-lactamase gene combined with a FRET-enabledsubstrate. Cells are loaded with an engineered fluorescent substratecontaining two fluoroprobes, coumarin and fluorescein. In the absence ofbeta-lactamase gene expression, the substrate molecule remains intact.In this state, excitation of the coumarin results in fluorescenceresonance energy transfer to the fluorescein moiety and emission ofgreen light. However, in the presence of beta-lactamase gene expression,the substrate is cleaved, separating the fluorophores, and disruptingenergy transfer. Excitation of the coumarin in the presence of enzymebeta-lactamase activity results in a blue fluorescence signal. Theresulting blue:green ratio provides a normalized reporter response.

Invitrogen (Carlsbad, Calif.) has exploited GeneBLAzer to build “Tango”assays that report drug binding to GPCRs. The Tango assay platform isbased upon ligand binding to GPCRs that triggers desensitization, aprocess mediated by the recruitment of intracellular arrestin proteinsto the activated receptor. As a result, the ligand-induced activation ofGPCRs may be assayed by monitoring the interaction of arrestin with thetest GPCR. A major advantage of this approach is that it does not dependon knowledge of the G-protein signaling specificity of the targetreceptor.

The target GPCR is fused at its intracellular C-terminus to an exogenoustranscription factor. Interposed between the receptor and thetranscription factor is a specific cleavage sequence for a non-nativeprotease. This chimeric receptor protein is expressed in a cell linecontaining the beta-lactamase reporter gene responsive to thetranscription factor. The cell line also expresses an arrestin-proteasefusion protein that recognizes and cleaves the site between the receptorand transcription factor. The assay is performed by adding a ligand tothe growing cells for a defined period and measuring the activity of thereporter gene. Activation of the reporter gene provides a quantifiablemeasurement of the degree of interaction between the target receptor andthe protease-tagged arrestin partner. Additionally, the Invitrogen Tangoassay is unaffected by other signaling pathways in the cell, thusproviding a highly selective readout of target receptor activation.

Protein complementation assays have been developed using (a)dihydrofolate reductase, (b) Green fluorescent protein and variants, (c)beta-lactamase, (d) luciferases, (e) aminogycosidephosphotransferase,and (0 CRE-recombinase to screen for drugs that modulate protein-proteininteractions, protein subcellular location, protein complexlocalization, and the association/dissociation of protein complexesMichnick, et. al., Drug Discov. 6:569-82 (2007), which is herebyincorporated by reference in its entirety.

For the whole-cell assays described below, in some cases a preliminaryin vitro screen using purified proteins as described in the nextsection, or a preliminary whole-cell assay at higher drug concentrationsmay be used to identify initial coferon ligands. In some of thedescriptions below, a beta-galactosidase system developed by DiscoveRxCorp. (Fremont, Calif.) is used, where the alpha-peptide withindependent affinity to the C-terminal enzyme acceptor protein (EA) isreferred to as ProLabel, and the alpha-peptide with weak to no affinityto EA is referred to as ProLink. Chemiluminescent or fluorescent signalgenerated by the reconstructed beta-galactosidase is determined asdescribed (Eglen review). Whole cell assays may not be as amenable tousing pooling techniques to screen for coferon pairs, thus the nanodroptechnology developed by Raindance Technologies (Lexington, Mass.) may bemore appropriate, (Leaman et. al, Nat. Methods 3(7): 541-43 (2006),which is hereby incorporated by reference in its entirety). Theadvantage of using whole cell assays is their immediate screen forcoferons that enter cells when targeting intracellular components. Thepotential disadvantage to whole-cell screens include identifyingcoferons that elicit the desired phenotype, but not through the intendedtarget. Carefully designed controls can reduce such false positives, andoccasionally, these “off-target” results will lead to drugs thatinfluence the process through alternative pathways.

K. Identifying Initial Sets of Coferon A Ligands that (Weakly) Bind tothe Target Protein

The gene for the target protein is linked to the coding sequence for theProLink alpha-complementing peptide. Upon activation, target proteinrecruits a second protein (i.e., GPCR recruits arrestin). The gene forthe second protein is linked to the gene for the EA acceptor protein.Linking of two proteins to each other may be accomplished by fusing theC terminus of one protein to the N-terminus of the second protein, withor without a flexible linker peptide, or alternatively using an inteinto splice the two proteins together, such that both proteins retainbiological function. Both of the above constructs are introduced intothe target cell. An HTS assay containing the target cells in each wellor nanodrop is set up, with from 1 to 10 or more coferon A variantligands and 1 or more coferon B generic ligands added to each well ornanodrop. A generic ligand may contain the scaffold and then thesimplest diversity element in all the diversity positions, for example,alanine if the diversity positions are filled with amino acid moieties.The number of variants will depend on the background level and hitlevel, determined experimentally. Likewise, the number of “generic”variants that can be tested within the same well or nanodrop may rangefrom 1 to 10 or more. The coferon dimer that best activates the targetprotein to recruit the second protein will best reconstruct thebeta-galactosidase ProLink and EA domains and give the strongestsignals. By using different sets of pools (i.e. rows vs. columns) alarge number of potential binders may be rapidly tested.

L. Identifying Optimized Coferon B Ligands that Pair With the InitialSets of Coferon A Ligands to Tightly Bind to the Target Protein

The gene for the target protein is linked to the coding sequence for theProLink alpha-complementing peptide. Upon activation, target proteinrecruits a second protein (e.g., GPCR recruits arrestin). The gene forthe second protein is linked to the gene for the EA acceptor protein.Linking of two proteins to each other may be accomplished by fusing theC terminus of one protein to the N-terminus of the second protein, withor without a flexible linker peptide or, alternatively, using an inteinto splice the two proteins together, such that both proteins retainbiological function. Both of the above constructs are introduced intothe target cell. An HTS assay containing the target cells in each wellor nanodrop is set up, with from 1 or more coferon A initially selectedligands and 1 to 10 or more coferon B ligands added to each well ornanodrop. The number of variants will depend on the background level andhit level, determined experimentally. The coferon dimer that bestactivates the target protein to recruit the second protein will bestreconstruct the beta-galactosidase ProLink and EA domains and give thestrongest signals. As above, when testing more than one coferon B ligandper well, use of different sets of pools (i.e. rows vs. columns) allow alarge number of potential binders to be rapidly tested.

In the procedures K and L above, the ProLink alpha-complementing peptidewas linked to a membrane bound receptor protein, which upon activationrecruits arrestin protein linked to the EA acceptor protein. Under theseconditions, agonist coferons may be identified by increasedbeta-galactosidase signal. Alternatively, the system may be turned on byaddition of a known agonist, and then antagonist coferons may beidentified by decreased beta-galactosidase signal. The above concept maybe expanded to include linking the target protein to the ProLabelalpha-complementing peptide. Upon activation, the target protein movesfrom the cellular membrane to the nucleus, where it can complement an EAacceptor protein that is localized to the nucleus. In the generalizedversion of this assay, binding of coferon to the target protein resultsin either an increase or decrease of reporter signal, cell growth orviability.

M. Identifying Coferon Dimers that Enhance Binding of Two Proteins WithWeak or no Binding Affinity to Each Other

The gene for target protein 1 is linked to the coding sequence for theProLink alpha-complementing peptide. The gene for target protein 2 islinked to the gene for the EA acceptor protein. Linking of two proteinsto each other may be accomplished by fusing the C terminus of oneprotein to the N-terminus of the second protein, with or without aflexible linker peptide or, alternatively, using an intein to splice thetwo proteins together, such that both proteins retain biologicalfunction. To identify a new weak binding partner to a given targetprotein, a yeast two-hybrid or other fish-bait protein complementationassay is set up, with both weak and strong hits identified. Both of theabove constructs are introduced into the target cell. A HTS assaycontaining the target cells in each well or nanodrop is set up, withfrom 1 to 10 or more coferon A and B dimer variants added to each wellor nanodrop. The number of variants will depend on the background leveland hit level, determined experimentally. The coferon dimer that bestenhance binding of the two proteins to each other will best reconstructthe beta-galactosidase ProLink and EA domains and give the strongestsignals. If necessary, candidate coferon A and B monomers that bindeither or both protein targets may be identified by a preliminary invitro screen (as in procedure A) or whole cell screen (as in procedureK).

N. Identifying Coferon Dimers that Further Enhance Binding of TwoProteins With Medium to Strong Binding Affinity to Each Other

The gene for target protein 1 or a mutant variant with weaker binding islinked to the coding sequence for the ProLink alpha-complementingpeptide. The gene for target protein 2 or a mutant variant with weakerbinding is linked to the gene for the EA acceptor protein. Linking oftwo proteins to each other may be accomplished by fusing the C terminusof one protein to the N-terminus of the second protein, with or withouta flexible linker peptide or, alternatively, using an intein to splicethe two proteins together, such that both proteins retain biologicalfunction. If one or both of the original proteins are used, they may beexpressed at a lower level. Often, some structural or sequenceinformation is available to guide alanine scanning or targetedmutagenesis to generate variants with the potential to bind weakly. Toidentify mutations that convert a strong binding partner into a weakbinding partner to a given target protein, a yeast two-hybrid or otherfish-bait protein complementation assay is set up to test mutantvariants, with both weak and strong hits identified. Both of the aboveconstructs are introduced into the target cell. A HTS assay containingthe target cells in each well or nanodrop is set up, with from 1 to 10or more coferon A and B dimer variants added to each well or nanodrop.The number of variants will depend on the background level and hitlevel, determined experimentally. The coferon dimer that best enhancebinding of the two proteins to each other will best reconstruct thebeta-galactosidase ProLink and EA domains and give the strongestsignals. The winning coferon dimer sets are then retested to determinewhich set enhances binding of the wild-type proteins to each other.

O. Identifying Coferon Dimers that Inhibit Binding of Two Proteins toEach Other

The gene for target protein 1 is linked to the coding sequence for theProLink alpha-complementing peptide. The gene for target protein 2 islinked to the gene for the EA acceptor protein. Linking of two proteinsto each other may be accomplished by fusing the C terminus of oneprotein to the N-terminus of the second protein, with or without aflexible linker peptide or, alternatively, using an intein to splice thetwo proteins together, such that both proteins retain biologicalfunction. Both of the above constructs are introduced into the targetcell. A HTS assay containing the target cells in each well or nanodropis set up, with from 1 to 10 or more coferon A and B dimer variantsadded to each well or nanodrop. The number of variants will depend onthe background level and hit level, determined experimentally. Thecoferon dimer that best inhibit binding of the two proteins to eachother will interfere with reconstructing the beta-galactosidase ProLinkand EA domains and give the weakest signals. If necessary, candidatecoferon A and B monomers that bind either protein targets in the absenceof the other protein may be identified by a preliminary in vitro screen(as in procedure A) or whole cell screen (as in procedure K).

P. Identifying Coferon Dimers that Inhibit Binding of Two Proteins toEach Other

The gene for target protein 1 is linked to the coding sequence for theProLabel alpha-complementing peptide. The ProLabel peptide sequence maybe modified to include a nuclear localization signal. The gene fortarget protein 2 is either currently or is modified to preferlocalization in the cytoplasm or at the cellular membrane. The gene forthe EA acceptor protein is modified to include a nuclear localizationsignal. These constructs are introduced into the target cell, and ifneeded, expression is adjusted such that under normal conditions bindingof target protein 1 (containing the ProLabel peptide) to target protein2 localizes the two proteins in the cytoplasm or at the cell membrane,thus preventing the ProLabel portion from entering the nucleus andcomplementing the EA acceptor protein, resulting in low or no backgroundlevel signal. Addition of from 1 to 10 or more coferon A and B dimervariants (in wells or nanodrops) that bind to target protein 2 in such away as to disrupt binding to target protein 1, allowing for transport ofthe ProLabel peptide (linked to target protein 1) to enter the nucleusand combine with the EA acceptor protein, and thus generating positivesignal. The number of variants will depend on the background level andhit level, determined experimentally. The coferon dimers that bestinhibit binding of the two proteins to each other will give thestrongest signals. If necessary, candidate coferon A and B monomers thatbind target protein 2 in the absence of the other protein may beidentified by a preliminary in vitro screen (as in procedure A) or wholecell screen (as in procedure K).

In this example, the ProLabel alpha-complementing peptide was localizedto the cytoplasm or cellular membrane by the two target proteins bindingeach other, while the EA acceptor protein was localized to the nucleus.The above concept may be expanded to include localization of theseproteins to the reverse or other compartments. In addition, in somecases binding of the two target proteins to each other will create abulky complex that would inhibit binding of the ProLabelalpha-complementing peptide to the EA acceptor protein, even if they arein the same compartment. The generalized version of this assay is onewhere binding of the two target proteins to each other squelches,inhibits, or occludes binding of the ProLabel alpha-complementingpeptide to the EA acceptor protein.

Q. Identifying Coferon Dimers that Inhibit Binding of Two Proteins toEach Other

The inverse of the above procedure may be performed using Target protein2 linked to the coding sequence for the ProLabel alpha-complementingpeptide, and Target protein 1 localized to the cytoplasm or at thecellular membrane. The gene for the EA acceptor protein is modified toinclude a nuclear localization signal. Addition of from 1 to 10 or morecoferon A and B dimer variants that bind to target protein 1 in such away as to disrupt binding to target protein 2, allowing for transport ofthe ProLabel peptide (linked to target protein 2) to enter the nucleusand combine with the EA acceptor protein, and thus generating positivesignal. Again, if necessary, candidate coferon A and B monomers thatbind target protein 1 in the absence of the other protein may beidentified by a preliminary in vitro screen (as in procedure A) or wholecell screen (as in procedure K).

R. Identifying Coferon Dimers that Inhibit Binding of Two Proteins toEach Other, Using a Helper Protein

The gene for target protein 1 is linked to the coding sequence for theProLink alpha-complementing peptide. The gene for target protein 2 islinked to the gene for the EA acceptor protein. Linking of two proteinsto each other may be accomplished by fusing the C terminus of oneprotein to the N-terminus of the second protein, with or without aflexible linker peptide or, alternatively, using an intein to splice thetwo proteins together, such that both proteins retain biologicalfunction. Both of the above constructs are introduced into the targetcell, which also produces a helper protein that may have weak or noaffinity to target protein 1. A HTS assay containing the target cells ineach well or nanodrop is set up, with from 1 to 10 or more coferon A andB dimer variants added to each well or nanodrop. The number of variantswill depend on the background level and hit level, determinedexperimentally. The coferon dimer that enhances binding of the helperprotein to target protein 1, and thus best inhibits binding of the twotarget proteins to each other will give the weakest signals. Ifnecessary, candidate coferon A and B monomers that enhance binding ofthe helper protein to target protein 1 in the absence of the otherprotein may be identified by a preliminary in vitro screen (as inprocedure C) or whole cell screen (as in procedure M).

S. Identifying Coferon Dimers that Inhibit Binding of Two Proteins toEach Other, Using a Helper Protein

The gene for target protein 1 is linked to the coding sequence for theProLabel alpha-complementing peptide. The ProLabel peptide sequence maybe modified to include a nuclear localization signal. The gene fortarget protein 2 is either currently or is modified to preferlocalization in the cytoplasm or at the cellular membrane. The gene forthe EA acceptor protein is modified to include a nuclear localizationsignal. These constructs are introduced into the target cell, which alsoproduces a helper protein that may have weak or no affinity to targetprotein 2. If needed, expression is adjusted such that under normalconditions binding of target protein 1 (containing the ProLabel peptide)to target protein 2 localizes the two proteins in the cytoplasm or atthe cell membrane, thus preventing the ProLabel portion from enteringthe nucleus and complementing the EA acceptor protein, resulting in lowor no background level signal. Addition of from 1 to 10 or more coferonA and B dimer variants (in wells or nanodrops) that enhance binding ofthe helper protein to target protein 2 in such a way as to disruptbinding to target protein 1, allowing for transport of the ProLabelpeptide (linked to target protein 1) to enter the nucleus and combinewith the EA acceptor protein, and thus generating positive signal. Thenumber of variants will depend on the background level and hit level,determined experimentally. The coferon dimers that enhances binding ofthe helper protein to target protein 2, and thus best inhibit binding ofthe two target proteins to each other will give the strongest signals.If necessary, candidate coferon A and B monomers that enhance binding ofthe helper protein to target protein 2 in the absence of the otherprotein may be identified by a preliminary in vitro screen (as inprocedure C) or whole cell screen (as in procedure M).

T. Identifying Coferon Dimers that Inhibit Binding of Two Proteins toEach Other, Using a Helper Protein

The inverse of the above procedure may be performed using target protein2 linked to the coding sequence for the ProLabel alpha-complementingpeptide, and target protein 1 localized to the cytoplasm or at thecellular membrane. The gene for the EA acceptor protein is modified toinclude a nuclear localization signal. Both of the above constructs areintroduced into the target cell, which also produces a helper proteinthat may have weak or no affinity to target protein 1. Addition of from1 to 10 or more coferon A and B dimer variants (in wells or nanodrops)that enhance binding of the helper protein to target protein 1 in such away as to disrupt binding to target protein 2, allowing for transport ofthe ProLabel peptide (linked to target protein 2) to enter the nucleusand combine with the EA acceptor protein, will generate a positivesignal. Again, if necessary, candidate Coferon A and B monomers thatenhance binding of the helper protein to target protein 1 in the absenceof the other protein may be identified by a preliminary in vitro screen(as in procedure C) or whole cell screen (as in procedure M).

Screening of Multimer Coferons

There are many proteins that function only when they assemble intomultimeric structures. The coferon design allows for expanding on themultivalency concept. One example is for inhibition of the heptamericprotective antigen which is responsible for anthrax toxicity. See FIG.27.

When considering coferon multimers, it should be recognized that thiscreates both unique opportunities in drug design, as well as uniquechallenges in screening for the best multimers. Multimeric coferons maybe used to bind to monomeric protein targets, targets comprised ofmultiple protein monomers or dimer subunits, or targets comprised ofmultiple different subunits. For example, consider a transportercomposed of 3 identical membrane subunits. A coferon drug could bedesigned wherein the linker element allows for self-assembly of 3molecules, each with the same diversity element “A”.

In the absence of assembled protein target the coferons bind reversiblyand weakly to each other. In addition, each individual coferon may haveweak binding to the transporter, but when combining four suchinteractions together, the tetrameric coferon structure may bindessentially irreversibly. See FIG. 28.

Alternatively, the coferon drug could be composed of two subunits (“A”and “B”), that assemble to form A-B heterodimers, and then continue toassemble to form a 6-membered circular structure of alternating A-Bcoferons. Each individual coferon may have weak binding to thetransporter, but when combining 6 such interactions together, thehexameric coferon structure may bind with the same avidity as full-sizedantibodies.

Assembly of linker elements into multimeric structures is discussed ingreater detail below (above), but there are some general concepts. Itmay be very difficult to identify the best binding coferon multimers ifmore than 3 of the ligands arise from diversity elements. Thus, onetheme is to screen for the best coferons under conditions where the samediversity element is connected two or more times to the dynamiccombinatorial chemistry element. Here the linker element will beconnected to two or more of the same drug molecule and may be in thesame geometry to cover two or more linker elements that would be presentin the final monomeric form of the coferon drug molecule.

The connection between the linker element and the diversity element mayalso vary. For example, when the same ligand binds to the same activesite in a dimer or tetrameric multimer of the same protein targetsubunit, the connector would most likely be a flexible (such as anethylene glycol) chain, to allow for each ligand to bind to an activesite, even though the active sites are on different faces of themultimeric protein. Alternatively, if the coferon is binding in a largegroove, then the linker element geometry may be critical, both ingenerating the overall shape of the multimeric scaffold, and inpositioning the diversity elements in the proper orientation.

Coferons, by virtue of their ability to bind to an extended surface areaof one or more macromolecules provide the opportunity to developenhanced versions of existing drugs, as well as entirely new classes ofinhibitors (See Table 1).

TABLE 1 Examples of Protein Families and Their Pharmacological TargetsEXAMPLES EXAMPLES OF ENDOGENOUS OF CURRENT CURRENT EXAMPLES OF TARGETTARGET LIGAND AGONISTS ANTAGONISTS DETECTION FAMILY EXAMPLE (MODULATORS)(ACTIVATORS) (INHIBITORS) ASSAYS G-PROTEIN β₂ epinephrine, albuterol,propranolol, HitHunter, COUPLED adrenergic norepinephrine salbutamol,butoxamine PathHunter RECEPTORS receptors terbutaline, (DiscoverX),salmeterol cAMP assay, Intracellular calcium flux, TANGO, GeneBlazer,ELISA, binding assays G-PROTEIN Muscarinic Acetylcholine Acetylcholine,Scopolamine, HitHunter, COUPLED receptors Pilocarpine atropine,PathHunter RECEPTORS ipratropium, (DiscoverX), caproctamine cAMP assay,Intracellular calcium flux, TANGO, GeneBlazer, ELISA, binding assaysG-PROTEIN H1 histamine Histamine diphenhydramine, HitHunter, COUPLEDhistamine doxylamine, PathHunter RECEPTORS receptor pyrilamine,(DiscoverX), brompheniramine, cAMP assay, chlorpheniramine,Intracellular Loratadine, calcium flux, Fexofenadine, TANGO, Cetrizine,GeneBlazer, Desoratadine ELISA, binding assays NUCLEAR Estrogen Estriol,17-beta- Tamoxifen, Hit-hunter RECEPTORS receptor⁽¹⁻³⁾ estrone,estradiol, ICI 164,384, (Discoverx), estradiol Chlorotrianisene,Keoxifene, reporter assays, Dienestrol, Mepitiostane TANGO, Fosfestrol,GeneBlazer, Diethylstilbestrol, ELISA, ligand Zeranol binding assays,VOLTAGE voltage- veratridine, tetrodotoxin, Intracellular ion GATED IONgated aconitine saxitoxin, flux assays CHANNELS sodium channels⁽⁴⁻⁶⁾VOLTAGE voltage- BAY K 8644, ω-conotoxin, Intracellular ion GATED IONgated CGP 28392 ω-agatoxins, flux assays CHANNELS calciumdihydropyridine, channels⁽⁷⁻⁹⁾ nifedipine LIGAND kainate glutamatekainic acid, CNQX, HitHunter, GATED ION receptor⁽¹⁰⁾ domoic acid,LY293558, PathHunter CHANNELS LY339434, LY294486 (DiscoverX), ATPA, cAMPassay, iodowillardiine, Intracellular ion (2S,4R)-4- flux, TANGO,methylglutamic GeneBlazer, acid ELISA, ligand binding assays, RECEPTORepidermal epidermal EGF, TGFa, PD153035, reporter assays, TYROSINEgrowth growth factor amphiregulin, anti-EGFR kinase assays, KINASESfactor betacellulin, antibody CO-IP, BRET, receptor epiregulin, C225,FRET, TANGO, (EGFR)^((11, 12)) neuregulins aeroplysinin- GeneBlazer, 1,AG18, HitHunter, AG82, AG99, PathHunter AG112, (DiscoverX), AG213, ELISAAG490, AG494, AG527, AG555, AG556 GROWTH Vascular VEGFR Ranibizumab,Hit-hunter FACTORS endothelial bevacizumab, (Discoverx), growthsunitinib, reporter assays, factor⁽¹³⁻¹⁶⁾ sorafenib, TANGO, axitinib,GeneBlazer, pazopanib, ELISA, ligand Naphthamides binding assays,PROTEASES Caspase⁽¹⁷⁾ granzyme B; Granzyme B, Z- caspase assays, caspasecaspase VAD(OMe)- apoptosis assays, FMK, Z- mitochondrial Dy, VAD-CHOCO-IP, BRET, FRET, TANGO, GeneBlazer, HitHunter, PathHunter (DiscoverX),ELISA PHOSPHATASES PP1^((18, 19)) phosphoserine/ calyculin A, proteintyrosine threonine nodularin, phosphatase residues tautomycin assay,CO-IP, BRET, FRET, TANGO, GeneBlazer, HitHunter, PathHunter (DiscoverX),ELISA PROTEIN ERK⁽²⁰⁻²²⁾ MEK AG126, kinase assay, CO- KINASES apigenin,Ste- IP, BRET, FRET, MPKKKPTPI reporter assays, QLNP-NH2, TANGO, H-GeneBlazer, GYGRKKRR HitHunter, QRRR-G- PathHunter MPKKKPTPI (DiscoverX)QLNP-NH2, PD98059, U0126, MISC Adenylate G proteins, bordetella NKY80,2′,3′- BRET, FRET, ENZYMES cyclase^((23, 24)) calcium pertussis,Dideoxyadenosine, calcium flux cholera toxin, 2′,5′- assays, cAMPforskolin Dideoxyadenosine, assays, TANGO, SQ22536, GeneBlazer, MDL-HitHunter, 12330A PathHunter (DiscoverX) MISCAcetylcholinesterase⁽²⁵⁻²⁷⁾ Caproctamine, Acetylcholinesterase ENZYMESMetrifonate, Assay, Amplex Physostigmine, Red, Ellman Galantamine,method, HPLC Dyflos, Neostigmine BIOACTIVE Ceramide⁽²⁸⁻³⁰⁾ sphingomyelinTNFα, Fas fumonisin B TLC lipid LIPIDS ligand, 1,25 charring, dihydroxydiacylglycerol vitamin D, kinase labeling in γ-interferon vitroCYTOKINES IL2⁽³¹⁻³⁷⁾ IL2R BAY 50- daclizumab, TANGO, 4798, P1-30,basiliximab, GeneBlazer, SP4206 SP4206 HitHunter, PathHunter(DiscoverX), IL2 dependent mouse CTLL cell line, ELISA MISC BCLXL⁽³⁸⁻⁴⁰⁾BAD BH3I-1, A- TANGO, PROTEINS 371191, GeneBlazer, ABT-737 HitHunter,PathHunter (DiscoverX), CO- IP, BRET, FRET, ELISA MISC p53⁽⁴¹⁻⁴⁴⁾ MDM2,JNK1- PRIMA-1, Pifithrin-α caspase assays, PROTEINS 3, ERK1-2, MIRA-1,apoptosis assays, p38 MAPK, RITA, mitochondrial Dy, ATR, ATM, CO-IP,BRET, Chk1, Chk2, FRET, TANGO, DNA-PK, GeneBlazer, CAK HitHunter,PathHunter (DiscoverX), ELISA MISC Tubulin^((27, 45, 46)) tubulinALB109564, kinase assay, CO- PROTEINS ABT-751, IP, BRET, FRET, D24851,reporter assays, D64131, TANGO, benomyl, GeneBlazer, β- estramustine,arrestin(DiscoverX LY290181 MISC β- L 1,10- Stagnant Amyloid PROTEINSamyloid⁽⁴⁷⁻⁵¹⁾ phenanthroline Fibril Formation derivatives, Assay,amyloid KLVFF, fibrillization assay LVFFA, Memoquin, SLF-CR MISCthymidylate raltitrexed, caspase assays, PROTEINS synthase⁽⁵²⁻⁵⁶⁾pemetrexed, apoptosis assays, nolatrexed, mitochondrial Dy, ZD9331,CO-IP, BRET, GS7904L, FRET, TANGO, fluorouracil GeneBlazer, HitHunter,PathHunter (DiscoverX), ELISA UBIQUITIN MDM2⁽⁵⁷⁻⁵⁹⁾ p53 trans-4-Iodo,TANGO, LIGASES 4′-boranyl- GeneBlazer, chalcone, HitHunter, Nutlins, MI-PathHunter 219, MI-63, (DiscoverX), CO- RITA, HLI98 IP, BRET, FRET,ELISA, reporter assay VIRAL HPV E2^((60, 61)) HPV E1 indandiones, E2displacement REGULATORS podophyllotoxin assay, TANGO, GeneBlazer,HitHunter, PathHunter (DiscoverX), CO- IP, BRET, FRET, ELISA, reporterassay BACTERIAL ZipA⁽⁶²⁾ FtsZ substituted 3- TANGO, CELL (2- GeneBlazer,DIVISION indolyl)piperidines, HitHunter, PROTEINS 2- PathHunter phenyl(DiscoverX), CO- indoles IP, BRET, FRET, ELISA, reporter assay,polarization competition assay, CYTOKINES TNF⁽⁶³⁾ TNFR infliximab,TANGO, adalimumab, GeneBlazer, etanercept HitHunter, PathHunter(DiscoverX), CO- IP, BRET, FRET, ELISA, SCAFFOLD JIP1^((64, 65)) JNKBI-78D3, TANGO, PROTEINS TIJIP GeneBlazer, HitHunter, PathHunter(DiscoverX), CO- IP, BRET, FRET, ELISA, kinase assay DNA PARP⁽⁶⁶⁻⁶⁹⁾INO-1001, TANGO, REPAIR AG014699, GeneBlazer, BS-201, HitHunter,AZD2281, PathHunter BS-401 (DiscoverX), CO- IP, BRET, FRET, ELISA,RIBOSOMES Antibiotics⁽⁷⁰⁾ ribosomes tetracyclins, cell death assay,macrolides, lincosamides, streptogramins HISTONE HDAC1⁽⁷¹⁻⁷³⁾suberoylanilide TANGO, DEACETYLASES hydroxamic GeneBlazer, acid,HitHunter, trichostatin PathHunter A, LBH589 (DiscoverX), CO- IP, BRET,FRET, ELISA, APOPTOSIS XIAP^((74,75)) SMAC/DIABLO, SM102-SM130 CO-IP,BRET, REGULATORS caspase 3, FRET, reporter caspase 7, assays, TANGO,caspase 9 GeneBlazer, HitHunter, PathHunter (DiscoverX), cell deathassays CHAPERONE Hsp90^((76,77)) Cdc37, Celastrol, CO-IP, BRET, PROTEINSsurvivin shepherdin FRET, reporter assays, TANGO, GeneBlazer, HitHunter,PathHunter (DiscoverX), SERINE/THREONINE mTOR^((78,79)) Raptor,Rapamycin, kinase assay, CO- PROTEIN mLST8/GβL caffeine, IP, BRET, FRET,KINASES farnesylthiosalicylic reporter assays, acid, TANGO, curcumin,GeneBlazer, temsirolimus, HitHunter, everolimus PathHunter (DiscoverX)SERINE/THREONINE- B-raf & B- K-ras PLX4720 kinase assay, CO- PROTEIN rafIP, BRET, FRET, KINASES V600E⁽⁸⁰⁾ reporter assays, TANGO, GeneBlazer,HitHunter, PathHunter (DiscoverX), CYCLIN CDK2^((81,82)) Cyclin A,Variolin, kinase assay, CO- DEPENDENT cyclin E Meriolin IP, BRET, FRET,KINASES reporter assays, TANGO, GeneBlazer, HitHunter, PathHunter(DiscoverX), GROWTH IGF-1R⁽⁸³⁾ IGFII PQIP CO-IP, BRET, FACTOR FRET,reporter RECEPTORS assays, TANGO, GeneBlazer, HitHunter, PathHunter(DiscoverX), PROTEASOME 20S^((84,85)) 19S Bortezomib, CO-IP, BRET,salinosporamide FRET, cell A, viabilityAll of the following citations are hereby incorporated by reference intheir entirety.

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At their most basic level, coferons may interfere or enhance proteinactivity where the substrate ranges in size from a medium tomacromolecule. For example, coferons may be designed to inhibitsequence-specific proteases, such as the caspases, which play a role inthe apoptotic pathway (See FIGS. 2.17A and B).

Coferons may be used to inhibit or facilitate protein-proteininteractions, including activating or inactivating a signaling pathway(FIGS. 2.17C, 2.19H and I). Coferons may activate signaling through morethan one mechanism. For example, the coferon may do more than link twoproteins together more tightly. It also further affects the conformationof the target protein so that it is more active compared to when the twoproteins are bound in the absence of coferon (FIG. 2.19H).Alternatively, coferons may shift the equilibrium to tighter binding sothat the numbers of complexes in the bound state is greater. In somecases, the coferon may act as a mimetic of a protein-proteininteraction, either activating or inactivating signaling from thattarget (FIGS. 2.18D-G).

To illustrate these concepts, consider the Wnt signaling pathway, whichis often disregulated in colon cancer. Wnt proteins bind to and activatethe Frizzled receptor, which in turn act via Dishevelled to suppress theactivity of GSK-3 β. Under normal conditions, GSK-3 β is part of acomplex with axin and APC, which binds β-catenin. However, whenDishevelled suppresses the activity of GSK-3 β, this prevents GSK-3 βfrom phosphorylating β-catenin, which therefore escapes degradation andaccumulates in the cytoplasm and in the nucleus. Once in the nucleus,β-catenin associates with Tcf/Lef transcription factor to drive theexpression of a variety of genes, such as Myc, which enable cellproliferation.

In this Wnt signaling pathway, coferons could be designed to: (i)Inhibit Wnt binding to Frizzled; (ii) Inhibit frizzled activation ofdisheveled; (iii) Inhibit Dishevelled inactivation of GSK-3 β; (iv)Enhance binding of β-catenin to Axin; and (v) Inhibit binding ofβ-catenin to Tcf/Lef.

In colon tumors, the APC gene is often truncated or reduced in copynumber or expression, thus it no longer binds β-catenin, liberatingβ-catenin to migrate into the nucleus. However, coferons designed toenhance binding of β-catenin to Axin, allow active GSK-3beta tophosphorylate β-catenin and send it down a path of degradation, thusavoiding proliferation and inhibiting tumor growth.

Some proteins, such as the tumor suppressor p53, are mutated in cancercells, causing them to unfold more easily and thus not functionproperly. Binding of a coferon across the surface of such a protein mayact as a molecular staple, keeping the domains or regions in the properconformation (FIG. 2.20). Likewise, some proteins undergo conformationalchanges, which may activate or deactivate enzymatic activity oradditional signaling. Coferons may be designed to bind one or the otherconformer more tightly, and thus act as an activator or inhibitor ofprotein function (FIG. 2.18).

There are examples in nature where a small molecule (FK506, rapamycin)uses a helper protein (FKBP) to create a composite surface that bindsthe target protein (calcineurin, FRAP) more tightly. This helper proteinmay be used to either recruit additional protein(s) or inhibit bindingof other proteins to the target protein. Coferons may be designed tomimic the role of FK506 to either enhance binding of a new protein tothe complex (FIG. 2.22O, FIGS. 2.24R-T), or inhibit binding of a newprotein to the complex (FIG. 2.23Q). In these examples (FIG. 2.22O,FIGS. 2.24R-S), the linker elements were designed to mimic the portionof FK506 that binds tightly to FKBP (“orange” protein), but many otherconfigurations may also be used.

Many proteins use protein interaction domains as modular units withintheir structure to achieve their desired functions. (See Table 2)

TABLE 2 Examples of Protein Domains EXAMPLE OF EXAMPLES APPROXIMATEPROTEIN OF K_(D) OF CONTAINING EXAMPLES OF KNOWN DETECTION BINDINGDOMAIN PARTNER DOMAIN INHIBITORS ASSAYS PARTNERS SH2 Phospho-tyrosineGrb2 Fmoc-Glu-Tyr-Aib- Surface 0.2-11 μM⁽⁵⁻¹⁰⁾ residues Asn-NH2; Ac-plasmon SpYVNVQ-NH2, resonance macrocycles, (SPR) STATTIC⁽¹⁻⁴⁾technology, FHA Phospho-threonine KIF13B 1-100 μM^((11, 12)) andphospho- tyrosine residues 14-3-3 Phospho-serine 14-3-3 R18⁽¹³⁾ 7 nM-20μM⁽¹⁴⁻¹⁶⁾ residues WW ligands containing Pin1 Zn(II) Dipicolylamine- 6μM-190 μM⁽¹⁸⁻²⁰⁾ PpxY, Proline-rich based artificial sequencesreceptors⁽¹⁷⁾ WD40 Apaf-1 1 μM⁽²¹⁾ MH2 phospho-serine SMAD2 240 nM⁽²²⁾residues BROMO acetylated lysine CBP 1 μM-4 mM⁽²³⁻²⁵⁾ residues UBAmono-, di-, tri-, and HHR23A 6 μM-2.35 mM⁽²⁶⁻²⁸⁾ tetra-ubiquitin PTBPhospho-tyrosine IRS-1 LSNPTX-NH2, PTB 160 nM-10 μM⁽³⁰⁻³³⁾ residues,Asn-Pro-X- LYASSNOAX-NH2, domain Tyr motifs LYASSNPAX-NH2⁽²⁹⁾ bindingassays SH3 Proline-rich peptides Grb2 Peptidimer-c, 1-500μM^((10, 35-37)) with consensus Pro- VPPPVPPRRR, X-X-Pro,(VPPPVPPRRR)2K)^((10, 34)) EVH1 FPxΦP motifs, ActA 10-50 μM⁽³⁸⁻⁴⁰⁾ PPxxFmotifs GYF proline-rich CDBP2 10-160 μM⁽⁴¹⁾ sequences, VHS TOM1 11-50μM⁽⁴²⁻⁴⁴⁾ PDZ PDZ, Val-COOH MNT1 NSC668036, FJ9^((45, 46)) 1-500μM⁽⁴⁷⁻⁵⁰⁾ PUF RNA PUM1 10-100 nM⁽⁵¹⁻⁵³⁾ TUBBY DNA, TULP1phosphotidylinositol SAM CNK 71 nM-1 μM⁽⁵⁴⁻⁵⁶⁾ DD DD FADD CARD CARDApaf-1 1.4 μM⁽⁵⁷⁾ PyD PyD Pyrin 4 μM⁽⁵⁸⁾ PB1 PB1 Bem1 4-500 nM⁽⁵⁹⁻⁶¹⁾BRCT BRCT BRCA1 113 nM-6 μM⁽⁶²⁻⁶⁶⁾ PH phosphatidylinositol- AKT1 NSC348900, 1.76 nM-350 μM^((30, 70-75)) 4,5-bisphosphate, perifosine, SH5,SH23, PI-3, 4-P2 or PI- SH24, SH25, ml14, 3,4,5-P3 ml15, ml16⁽⁶⁷⁻⁶⁹⁾FYVE Phosphatidylinositol SARA 50 nM-140 μM 3-phosphate, zinc C1 phorbolesters, PKC 0.58-800 nM⁽⁷⁶⁻⁷⁹⁾ diacylglycerol isoforms FERM PI(3)P,PI(4)P, PTLP1 200 nM-30 μM⁽⁸⁰⁻⁸²⁾ PI(5)P, IP3, C2 Calcium, acidic Nedd4250 nM-94 μM⁽⁸³⁻⁸⁵⁾ phospholipids PX PI(3,4)P2, PI(3)P, CISK 1.8 nM-50μM^((36, 86, 87)) PI(3,5)P2, PI(4)P, PI(5)P, PI(3,4,5)P3, PI(4,5)P2 ENTHPtdIns(4,5)P2, Epsin1 98 nM-1 μM⁽⁸⁸⁻⁹⁰⁾ PtdIns(1,4,5)P3, PI(3,4)P2;PI(3,5)P2All of the following citations are hereby incorporated by reference intheir entirety.

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SH2 domains are found in    proteins that act as, or play a role in: adaptors, scaffolds,    kinases, phosphatases, ras signalling, transcription,    ubiquitination, cytoskeletal regulation, signal regulation, and    phospholipid second messenger signaling. As another example, SH3    domains bind peptide loops with the motif RXXK or PXXP. Many    proteins have both SH2 and SH3 domains, which act as “receptors” to    bind one or more protein partners. Coferons may be designed to    inhibit binding of a phosphotyrosine protein to its cognate SH2    domain. Alternatively, coferons may be designed so one ligand binds    one motif (i.e. SH2), and a second ligand binds a second motif (i.e.    SH3), either on the same or different proteins.

Many large proteins or macromolecular complexes (such as ribosomes—seebelow, tubulin filaments) have multiple binding sites with known druginhibitors. Coferons may be used to bring together two previous drugs onthe same target to: (i) bind the target with higher affinity; (ii)exhibit a stronger inhibition than either drug alone; (iii) exhibitgreater activation than either drug alone; or (iv) create a bindingentity covering a larger surface area of the target, making it harderfor the organism/cell/virus to develop resistance to the drug via pointmutations.

Coferons may be used to create bifunctional drugs that bind to the sametarget, for example, protein receptor tyrosine kinases. One ligand wouldbind to the ATP binding site, while the other mimics the auto-inhibitingpeptide. These two ligands would be attached to separate coferons, whichwhen brought into the proper proximity by linker element binding, wouldlock down into both binding pockets and bind the receptor kinase withexcellent specificity. This approach would overcome limitations ofearlier inhibitor designs that bind only to one pocket and,consequently, lack either proper specificity, or sufficient bindingaffinity to be effective drugs in vivo.

Combining multiple known drugs using coferons may generate new classesof agonists or antagonists for: protein kinases, calcium channelproteins, muscarinic receptors (antagonists), beta-2 adrenergic receptor(agonist), sodium channel drugs, and H1 histamine receptor(antagonists). See Table 1. Receptor proteins provide multipleopportunities for coferon design to inhibit, activate, dampen, oramplify signals (FIG. 2.28 and FIG. 2.29).

Many proteins act as dimers. Homodimer coferons could act as agonists tohelp keep two receptors close enough for auto-phosphorylation andactivation (FIG. 2.25 B2). Homodimers could also act as antagonists, bypreventing two receptors from undergoing auto-phosphorylation (FIG. 2.26C2). Coferon heterodimers may also act to dampen (FIG. 2.26 D2) oramplify (FIG. 2.27 E2) ligand directed signaling.

Use of coferon homodimers may also help inhibit dimer enzymes byblocking both ligand-binding sites simultaneously (FIG. 2.30 A3). Suchhomodimer, homotetramer, heterotetramer, hexamer, and other multimercoferons may have PEG linkers or other spacers to the linker elements,allowing for binding two sites that are several nanometers apart (FIGS.2.30 B3 and C3, FIGS. 2.31 D3-F3). They may use linker elements thatbind to each other with minimal or no added help from the ligand bindingevents.

Many proteins have allosteric sites to either activate or inhibitenzymatic activity. Such sites are generally too distant from the activesite to allow for a traditional small molecule drug to bind to bothsites simultaneously. However, heterodimer coferons composed of ligandsthat bind into both the allosteric and either adjacent or active siteregions would be potent activators or inhibitors.

Microtubulins play a key role during mitosis and differentiation, andthus are targeted in treating tumors. Microtubulins are composed of twosubunits, alpha and beta tubulin that are in a dynamic instabilityeither assembling or disassembling during the cell cycle. Duringmitosis, the rates of both assembly and disassembly are increased sothat the chromosomes can capture the microtubules forming the mitoticspindle. During differentiation, microtubule-associated proteins helpstabilize the filaments, thus allowing cellular cytoplasm to organize.Vinca alkaloid anticancer agents such as vincristine and vinblastine arecytotoxic by disrupting microtubules, while taxanes such as palitaxeland docetaxel stabilize microtubules, and thus may nudge tumor cellstoward differentiation. Coferon pairs composed of one or two tubulinligands may have enhanced antitumor activity (see FIG. 2.32).

Many neurodegenerative diseases arise due to misfolding of proteins thataggregate to form plaques. For example, Alzheimer's disease arises dueto plaques composed of amyloid beta-peptide. Since coferons assemble atthe target site, there is an opportunity to design coferons small enoughto traverse the blood-brain barrier, yet large enough to combine on thesurface of amyloid beta-peptide monomers and inhibit formation ofoligomers and ultimately amyloid fibrils (FIG. 2.33).

Some linker element designs may allow linker elements to bind to eachother with minimal or no added binding help from the diversity elements.Such designs include linker elements that bind to each other with theaid of a metal cofactor (see below). These designs expand the potentialuses of coferons.

As another example of irreversible association within a cell, onecoferon may have a disulfide group beta to a primary amine, while theother may have a ketone group. In the blood stream or in non-cancerouscells, the two coferons may associate through forming a Schiff basebetween the amine and the ketone group. However, upon entering cancercells, the disulfide is reduced to a thiol, which may then act inconcert with the primary amine to create a thiazolidine linker. Suchdimer coferons may be used to bring two target proteins into closeproximity.

Coferons using linker elements that bind to each other with minimal orno added help from the target binding event may be used to generatebifunctional drugs to different targets. Such drugs would concentratetwo cancer-fighting ligands into the same cancer cell. This approach isalso being used with HIV drugs.

Such coferons may also be used to create trap-door drugs. One coferonwould be designed to bind to a target that is found in abundance in thetarget cancer cell, but not so frequently in normal cells. This coferonwould be administered first to the patient. Subsequently, a secondcoferon with known drug moiety would be administered. The second coferonenters most cells, but then is preferentially trapped in target cancercells. This approach may need to use coferons with almost irreversiblelinkages between linker elements.

The trap-door concept may be used in reverse to clog drug export pumps,many of which are responsible for resistance to chemotherapy. Coferonsare designed to enter cells as monomers. One of the diversity elementsis a substrate for export. However, when the first coferon covalentlyattaches to second coferon, this creates a plug to clog the export pump.Such a coferon “plug” would be combined with a traditional cancer drug.This concept is similar to augmentin (amoxicillin clavulanate), wherethe clavulanic acid inhibits beta-lactamase.

The above examples emphasize the ability of coferons to inhibit,modulate, or activate protein-protein interactions. Coferons may alsoinhibit, modulate, or activate other major worlds of macromoleculeinteractions. For example, coferons may be used to tuneprotein-protein-nucleic acid interactions when transcription factorsbind to dsDNA, or proteins that bind to RNA (e.g. ribosome). These couldbe every bit as significant wherein one targets the protein and thenucleic acid interaction by coferons. Many proteins undergomodifications (i.e. phosphorylation, acetylation, methylation,sumolation, prenylation, and ubiquitination), where these modificationsallow for signaling, transport, or degradation through additionalprotein interactions. All of these processes may be inhibited oractivated by judiciously designed coferons. Larger modifications, suchas synthesis of glycoproteins provide the potential for coferonsblocking interactions when proteins bind to the carbohydrate moieties.

Many proteins have signals to move them to various compartments ormacromolecular structures.

Coferons may be used to bring together two proteins to either accelerateor inhibit movement of the two proteins to the: (i) membrane, (ii)cytoplasm, (iii) mitochondria, (iv) lysosome, (v) proteosome, (vi)golgi, (vii) endoplasmic reticulum, (viii) extracellular space, (ix)nucleus, (x) cellular filaments or scaffolding, or (xi) otherintracellular or extracellular compartment, cellular structure, orspace.

Coferons provide a unique opportunity for targeted entry into cancercells. In the most direct form, folic acid is used as both the linkerelement and a means to transport the drug moiety into cancer cells. Thefolate transporter is found over-expressed in many cancers andespecially in metastatic cancer cells. Thus, the folate transporterhelps concentrate the drug molecule within cancer cells. Folic acid andderivatives are very “sticky” and tend to associate with each other.This association may be enhanced by addition of appropriate reactivegroups (preferably, those forming reversible covalent bonds) to the twofolic acid linker elements.

An alternative use of folic acid is as a transporter of a coferonprecursor into the cells. Here, the folic acid group is linked to thecoferon via a disulfide bond. Glutathione levels are 1.000-fold higherin tumor cells than in the blood. Inactive form of thiol-containingcoferon is internalized, then opened by glutathione, brought intoproximity with it's coferon pair (also activated by glutathione). Thereleased thiol groups are then available to participate in crosslinkingreactions when two coferons come together ultimately leading to celldeath. This approach has the advantage that the coferon drug moleculesare in an inactive precursor form in the blood stream as well as normalcells, but are activated upon entering cancer cells.

Potential transporters of coferon or coferon-cofactors include: glucosetransporter, taurine transporter, cationic amino acids transporter,organic anion transporter, proline transporter, monoamine transporter,Anion exchange transporter, folate transporter, monocarboxylic acidtransporter, Zn transporter, amino acid transporter, Na dependentvitamin transporter, fatty acid transporter, nucleoside transporter, andproton-coupled divalent metal ion transporter.

Subunits of the above transporters are overexpressed in both primary andmetastatic colon tumors. Use of transporters or receptors may provide asecond life for existing drugs. An existing drug is attached to a linkerelement that binds its pair independent of target to create the firstcoferon. The second coferon has affinity to transporter specific to thetarget organ or target tumor, specific to a receptor protein on the cellsurface or even to a cytoplasmic protein, any one of which may help pullthe drug on the first coferon into the desired cells. Some uptakesystems bring the solute into an endosome where it is released from thetransporter (for example by a change in pH). In some of these cases, thedrug molecule may still need to cross a membrane. One advantage ofcoferons is that the linker element portion may be modified, for examplemade more lipophilic, such that the entire coferon is more easilytransported into the target cell.

For the folate transporter, the folic acid may be used as a linkerelement. For the zinc or other divalent metal ion transporter, the zinccould be a co-factor to assist two linker element in binding with eachother. Zinc is uniquely suited to be a cofactor as it is generally anon-toxic metal ion that may be ingested in large quantities, up to 40mg per day for adults for up to a week. Thus, a cancer patient would beable to preload the cancer cells with zinc, which would then help trapcoferon drugs that bind each other through linker elements that chelatea zinc ion as a cofactor. Further, bidentate organic molecules that bindzinc depend on a very precise geometric and electronic configuration ofthe ligand structure, thus such coferon drugs would be less likely tobind other divalent cations nonspecifically.

Cancer cells provide multiple opportunities to take advantage of theunique properties of coferons. For example, coferon pairs may besynthesized to contain spatially separated ketone and a disulfide grouptwo carbons from a primary or secondary amine. When screening forsuitable diversity elements in vitro, the disulfide group remainsoxidized. Coferon pairs can form via a reversible imine (primary amine)or imminium ion (secondary amine) formation. Dynamic combinatorialchemistry is used to select the best diversity elements. When thewinning pair of coferons is introduced into the patient, the coferonsremain as monomers (occasionally associating to form dimers) until theyenter the cell. The disulfide bond is reduced by internal glutathione,and then the liberated thiol group on the coferon can now react with theimine or imminium ion to form an irreversible thiazolidine link betweenthe two coferon pairs. Judicious choice of the linker element design candrive the reaction forward only inside cancer cells containing thedesired target.

Additional approaches to unmasking reactive groups of coferons uponentering target cells include but are not limited to use of esterases tocleave esters and liberate a reactive alcohol group, and peptidases toliberate a reactive amino group.

Coferons as Multivalent Drugs Against Bacteria.

There are a number of antibiotics that inhibit or interfere with properribosome function. Aminoglycosides (gentamicin, tobramycin, amikacin,kanamycin, neomycin, paromomycin) induce formation of aberrant,nonfunctional complexes, as well as causing misreading of the mRNA. In asecond mechanism, some aminoglycodies also prevent the transfer ofpeptidyl tRNA from the A-site to the P-site, thus preventing elongationof the polypeptide chain. Aminoglycosides bind irreversibly to specificribosomal proteins. Streptomycin binds S12 in 30S subunit, while othersbind to the L6 protein of the 50S subunit.

Tetracyclines (tetracycline, minocycline, doxycycline, demeclocycline)binds reversibly to 30S ribosome.

Inhibits binding of aminoacyl tRNA into the A site of the bacterialribosome. Chloramphenicol inhibits peptide bond formation by binding toa peptidyltransferase enzyme on the 50S ribosome.

Macrolides (erythromycin, azithromycin, clarithromycin, dirithromycin)are large lactone ring compounds that bind reversibly to the 50Sribosomes and impair the peptidyltransferase reaction (i.e. preventforming a peptide bond between the amino acids), or translocation (i.e.preventing transfer of the peptidyl tRNA from the A-site to the P-site),or both.

Oxazolidinones (Linezolid) bind to the 50S subunit and interfere withformation of the mRNA, f-met-tRNA and 50S subunit complex. Lincosamides(clindamycin) also inhibits protein synthesis by binding to the 50Sribosome.

Coferon dimers containing one each of the above drugs from two differentbinding regions as the ligands may show greater biological activity thanthe monomers. This may be especially true if the drugs bindsynergistically, and are kept in the approximate proper orientation bythe linker element tether. Such drugs may also stay within cells longer,allowing for more intermittent dosing of the drug. Finally, it may bemore difficult for the bacteria to mutate both monomeric drug bindingsites simultaneously.

Coferons as Drugs Against Rapidly Evolving Viruses.

RNA viruses are a constant public health threat as their rapidlyevolving genomes have outwitted repeated attempts to generateneutralizing antibodies or vaccines. The last 20 years has seen enormousstrides in the synthesis of inhibitors to various viral proteins, suchas proteases and reverse transcriptase. Nevertheless, in time virusesescape these drugs through mutational selection to resistance. Coferonsprovide two unique opportunities to inhibit RNA viruses. Resistantvariants for many existing drugs are now known, and thus coferons may bescreened against both sensitive and resistant variants, allowing forselection of the winning families or clades of coferon monomers. Use ofa limited number of each family member (for example 10 each for coferon“A” and coferon “B”) allows for addition of a “therapeutic cocktail”where the protein target selects the tightest binding pair (which willbe 10% of the total molecules) and thus selects for its own strongestinhibitor. A second opportunity arises from viral protein interactionswith a human host protein, and this interaction may be disrupted byidentifying coferons that bind to the host protein, or bind and recruita second protein to the host protein, and thus either directly orindirectly inhibit binding of the viral protein to the host protein.Below are some examples based on HIV.

HIV Protease

From structural work and alanine scanning mutagenesis studies, thecontact points for HIV protease and its substrates are determined. Then,families of “A” and “B” coferons are designed, such that the combinationof A+B provide enough structural space to allow binding to mutationalvariations in the target HIV protease, thus achieving desired inhibitionof said protease. Since coferons A+B bind reversibly, dynamiccombinatorial chemistry will assure that each protease variant binds thetightest inhibitor combination.

HIV Entry

HIV entry into cells depends on binding to the CCR5 receptor. Whileattempts to make vaccines to the HIV envelope protein have beenunsuccessful, coferons could be designed to bind to the CCR5 receptor,either as a dimer, tetramer, or recruiting another protein to CCR5, thusblocking the HIV from binding to the same receptor.

HIV Reverse Transcriptase

Traditional reverse transcriptase inhibitors are based on nucleotideanalogues. However, resistant variant reverse transcription easilyarises. Coferons could be more effective in inhibiting this enzyme bydesigning a family of nucleotide analogs “A” which bind both “wild-type”and different drug resistant variations of HIV reverse transcriptase,and a family of second drugs “B” that bind the HIV RT elsewhere.Combining coferons A+B provides enough structural space to allow bindingto mutational variations in the target HIV reverse transcriptase, whilestill inhibiting its activity.

HIV Vif Protein

Human cellular protein A3G sabotages HIV by dramatically mutating itsgenes. HIV Vif protein interferes with this process. One approach is touse coferons to generate a mimetic decoy of A3G, such that the HIV Vifprotein binds the coferons instead of the A3G protein. A second approachis to use coferons to bind to A3G, or bind and recruit another cellularprotein to A3G, thus blocking Vif binding to A3G. Since A3G is a humanprotein, and not undergoing the same mutational drift as the HIV Vifprotein, it is easier to design coferons that either mimic, or bind toA3G.

HIV Integrase

HIV integrase, with the help of the human cellular protein LEDGF,integrates the ds DNA copy of the virus into the human genome. Coferonsmay be selected to interfere with HIV integrase activity, as well asintegrase binding to LEDGF. As above, since LEDGF is a human protein,and not undergoing the same mutational drift as the HIV integraseprotein, it is easier to design coferons that either mimic, or bind toLEDGF.

Coferons Containing Nucleic Acid and Oligonucleotide Analogues Ligands.

Antisense and siRNA studies have provided an excellent tool for knockingdown specific transcripts in individual cells. To date, with a fewexceptions, this promise has not been born out in whole mammalianorganisms.

Coferons provide an opportunity to overcome problems related totransport of RNA or RNA analogues into cells. Coferons can combine twooligonucleotide ligands through their linker elements. Thus, currentapproaches that are being used to transport RNA or oligonucleotideanalogues into cells may become more efficient if the transportoligonucleotide is half the size, i.e. reducing from a size of 19 to 22bases, to two oligonucleotides ranging from approximately 9 to 12 basesand containing linker elements, allowing for their assembly on thetarget to full length RNA with the appropriate biological activity (seeFIG. 2.34). For siRNA, double-stranded RNA of sizes 21 to 27 bases havebeen used, again allowing for assembly of smaller oligonucleotides aftertransport. Efforts to transport nucleic acid and analogue macromoleculesinto the cell include using lipophilic carriers, and attaching them toother molecules (such as folic acid) that are actively transported intotarget cells (such as cancer cells, which often over-produce the folicacid transporter.

Coferons may also help transport RNA, or oligonucleotide analogues intocell using two new formulations herein termed “Lipocoferons” and“Aminoglycoferons” (see FIG. 2.35).

“Lipocoferons” consist of 2 or 3 amino groups spaced to bind to thenegatively charged nucleic acid backbone. Lipocoferons have ahydrophobic face to help transport nucleic acid fragment into the targetcell. In one variation, Lipocoferons have free thiol groups allowing thelipocoferons to form multimers when binding to the nucleic acidfragment, but then once inside the cell, internal glutathione reducesthose thiol linkages releasing the lipocoferons from the RNA oroligonucleotide analogues.

“Aminoglycoferons” are based on known aminoglycosides that bind RNA. Inone variation, they are designed to allow for linking together usingthiol linkages. They can bind to RNA and help transport nucleic acidfragment into the target cell. Once inside the cell, internalglutathione reduces those thiol linkages releasing the aminoglycoferonsfrom the RNA or oligonucleotide analogues.

Once the nucleic acid fragments or nucleotide analogue fragments areinside the cell, they will bind to RNA sequences. The coferon designprovides an opportunity to dramatically improve the efficacy ofantisense type drugs by using shorter RNA or oligonucleotide analogues,preferably ranging from 9 to 12 bases, whose sequences are complementaryto the upstream half and downstream half of the target RNA sequence. Inthe preferred embodiment, each Coferon contains a linker element, suchthat when the two portions bind to their target with perfectcomplementarity at the junction, the linker elements are positioned tojoin via either covalent or non-covalent binding. Such joining increasesthe binding energy and avidity to the correct target, allowing for theappropriate biological activity.

Oligonucleotides with lengths of 9 to 12 bases bind to complementarysequences with melting temperatures ranging from on average 27° C. (for9-mers) to 37° C. (for 12-mers), although Tm's for specificoligonucleotides of this size may vary from a low of about 18° C. to ahigh of about 48° C. Under physiological conditions and at humantemperatures of 37° C., individual oligonucleotides will be inequilibrium between binding to the desired RNA target, other RNA, andnot binding to the target. Coferons with nucleic acid or nucleotideligands of a given length may be modified in the backbone (i.e.2′-O-methyl, PNA, LNA), base (i.e. 5-propynyl C) and/or with additionalgroups (i.e. hydrophobic groups, minor-groove binding moieties) toincrease the overall binding to an RNA target at a given temperature.Such an increase will shift the equilibrium to greater binding at boththe desired target site as well as at off-target sites. However, oncetwo coferon-nucleic acid/analogue bind adjacent to each other on thecorrect target, they become linked, thus stabilizing and greatlyincreasing the affinity of the coferon dimer to only the correct target.The Tm's for coferon dimers ranging in length from 18 to 24 bases willbe on average from 54° C. to 72° C., although Tm's for specificoligonucleotides of this size may vary from a low of about 36° C. to ahigh of about 92° C. Thus, the coferon dimers bind to the correctsequence with 100 to 1.000-fold higher avidity then either monomeralone, and furthermore, once assembled on the correct target will notdissociate at any appreciable rate.

Coferon-nucleic acid/analogue drugs thus have a tremendous advantageover traditional antisense oligonucleotides in the fact that the coferonmonomer moieties are substantially smaller, and therefore are easilydissociated from binding to off target sites—in contrast to thefull-length antisense oligonucleotides, which have been shown to havesubstantial off-target effects in certain cases.

Coferon-nucleic acid/analogue drugs may also be designed to interactwith the RNA degradation machinery (See FIG. 2.36). For example, RNAcoferons may be designed with antisense coferons of 12 and 15 bases inlength, and the sense oligonucleotides being of 15 and 10 nucleotidelength respectively. Once transported into cells, hybridization of theseoligonucleotides would allow for assembly of an antisenseoligonucleotide of 27 nucleotides held together by the linker elements,and a “split” sense strand of 25 nucleotides. This structure would be asubstrate for Dicer and suitable for uptake by the RISC complexresulting in an enzyme complex that would catalytically degrade mRNAcomplementary to the antisense RNA composed of two coferon-nucleicacid/analogue drugs. Other configurations of coferons could interferewith other biological activity, including interfering with translation(siRNA), degrading or inhibiting transcripts (miRNA), or enhancingtranscription (aRNA).

The approach for Coferon-nucleic acid/analogue drugs may be expanded toinclude (i) linking more than two coferon-nucleic acid/analoguestogether, (ii) using two coferons, one composed of an aminoglycocideknown to bind bacterial or fungal ribosome, with a secondcoferon-nucleic acid/analogues designed to bind an adjacent region ofribosomal RNA in the target, and (iii) using two coferons, one composedof a coferon-nucleic acid/analogue that binds a ribozyme or RNA target,with the second coferon composed of a small molecule ligand that bindsat an adjacent position in the ribozyme or RNA target.

Screening Multimer Coferons.

For multimer coferon screening, one starts with diversity libraries andknown ligands or group of ligands in the following formats: (i) on abead or solid support with monomer diversity element defined by positionor bar-code encryption of particle, (ii) same as previous, except nowtwo or more identical diversity elements are on the same coferon anddefined by position or bar-code encryption of particle, (iii) insolution (off the bead) with monomer diversity element defined by anencoded DNA element, (iv) same as previous, except now two or moreidentical diversity elements are on the same coferon and defined by anencoded DNA element, (v) one or more known ligands on a bead or solidsupport, with ligand defined by position or bar-code encryption ofparticle, and (iv) one or more known ligands in solution, with liganddefined by position or encoded DNA element.

The advantage of working with coferon libraries attached to beads isthat each bead contains multiple copies of the identical ligand. Thisproperty helps identify the strongest affinity ligand combinations bythe intensity of fluorescently labeled entity captured (i.e. protein orother ligand). When synthesizing coferons on a solid support, thespacing of individual coferons should be sufficient to avoid binding thetarget by multiple copies of the same coferon. This potential artifacthas led to “identification” of target binding peptides through phage orother display technologies, only to find the original effect disappearswhen using individual in-solution peptides. This artifact may be avoidedor mostly limited by the following approaches:

(i). Use of limited loading of a reactive group on the bead or solidsurface—such that on average only 1 out of every 100 adducts is closeenough to the next so that both can bind to the same target.

(ii). Use of Streptavidin coated (magnetic) beads, followed by loadingthe reactive group on biotin. In a slightly more sophisticated versionof this, the bead is coated with biotin on very short spacers, such thatonly one group can bind a streptavidin. A reactive group containingthree biotin “hooks” is added. Once one catches the streptavidin, theother two will bind the remaining sites. Thus, on average 1 reactivegroup is bound per streptavidin tetramer.

The advantage of working with coferon libraries encoded by DNA is thatselected coferons may be amplified by using their DNA to template asecond round of diversity element synthesis. This allows forevolutionary principles to be used in selecting the best coferons.Finally, we also consider that diversity elements may be synthesized onbeads and then released without any encoded DNA element. When used underconditions where sufficient coferon binds to both protein target andbeads, the structure of the diversity element may be identified ornarrowed down using mass spectroscopy. This approach has the advantageof obviating the need to attach an encoded DNA element to the coferon,which may influence the binding event. If the number of coferons testedcan be limited by in silico pre-screening of potential diversityelements binding to a known 3-dimensional structure of the target, thenuse of mass spectroscopy to identify the winning coferon from in vitroscreening becomes a highly efficient process.

Screening multimer coferons binding to the target is based on liberatingthe process of screening for the best diversity elements from theprocess of identifying the best linker element design to be used in thefinal coferon drug. During the screening process, two or more identicaldiversity elements may be tethered together. The tethering mayrecapitulate the precise geometry of the linker elements and diversityelements by simply tethering two linker elements together, or thetethering may approximate the geometry of the diversity elements andreplace the linker elements altogether. Once the optimal diversityelements have been identified, the final coferons are re-synthesized asmonomer subunits containing the correct linker element and the selecteddiversity element.

Derivatives based on mother-child linker elements (M-Coferons)M-coferons are coferons that possess a single “mother” linker elementcapable of linking to multiple “child” linker elements from C-coferons.The M,C coferon system is designed to target protein multimers,especially those that contain a channel or cavity. Examples wouldinclude transporters (p-glycoprotein, polyamine transporter),proteasomes, viral protein coats, biomolecular machines. This isillustrated in FIG. 29.

An example of the M,C coferon system which utilizes a disaccharide(lactose in the following example) as the M-coferon and a boronate asthe C-coferon. Disaccharides are of particular interest since there arespecific transporters for them, e.g. galactose receptors are found onthe surface of cancer cells.

Non-saccharide polyols may also serve as M-coferons as shown in theexample below.

Screening for A-B-C Target Binding Coferons

Two types of geometries for these trimeric coferons are envisioned. Thefirst has both the A and C linker elements binding with the B linkerelement. The simplest version of such linker elements would containaromatic rings that stack on each other. The second geometry has Abinding to B, B to C, and C to A. This may be achieved using linkerelements that incorporate a cyclopropane scaffold to achieve 60° C.angles between the three linking groups. Each cyclopropane would bedisubstituted with groups suitable for making reversible linkages. Forexample, both A and B may contain an aldehyde and sulfhydryl in thetrans stereochemistry, and form a disulfide bond. The aldehyde groupswould both be on the same side of the cyclopropane's and would thus beable to link up with a linker element containing cis diols. Lessconstrained cyclopentane structures would also be compatible with thisselection linking groups for generating tri-meric coferons.

A variation of this theme is the A-B-A or A-A-B target binding coferon.Here one of the diversity elements is repeated twice, but addsadditional binding energy.

A first version of a screen (similar to that in FIG. 9) uses diversityelements on both beads and in solution. Fluorescently labeled targetprotein is added to the beads (containing “A” coferon) and in-solution“B” coferons, and after a suitable period of panning, wells containingfluorescently labeled beads are identified. If the beads are in a singlechamber, the barcode is identified for the fluorescently labeled beads.In both cases, the in-solution “B” coferons are identified by PCRamplification and sequencing of the DNA tags, or by mass spectroscopy ifthere is no bar-code. This process will identify many “A“−”B” coferonpairs that bind to the target. These winning pairs are then used to fishout the “C” coferon. This may be accomplished by synthesizing either adimer, or individual monomers of “A” and “B” coferons in solution,adding to “C” coferons on beads, and fluorescently labeled targetprotein, panning under more stringent conditions and identifying wellscontaining fluorescently labeled beads. The “A” and “B” coferons boundto each fluorescent bead are identified either by PCR amplification andsequencing of the DNA tags, or by mass spectroscopy if there is nobarcode.

In a variation of this theme, the “A” coferon on the bead is synthesizedwith two identical diversity elements. This allows both of them toprovide an increased binding affinity. A simple way of accomplishingthis is to increase the loading of the “A” coferon on the solid support,such that many can readily dimerize. Those beads containing “A” coferonsthat are close enough to form dimers, and that provide a selectiveadvantage in binding the (fluorescently) labeled target protein willshow the highest label (i.e. fluorescence). Using this approach theoverall geometry of a tri-meric coferon would be maintained, with thesecond “A” coferon acting as a placeholder. The second A coferon couldbe replaced by a “C” coferon in an additional screen, although if theinitial binding energy is strong enough, the final drug could becomposed of two identical “A” subunits, and one “B” subunit. This ideamay also be extended to identify tetrameric target binding coferons,composed of two identical “A” subunits and two identical “B” subunits.

The PCR amplified tags may also be used to re-synthesize the diversityelements, such that evolutionary principles are used to select thewinning sets of coferons. Alternatively, each of the top ligands may bere-synthesized and can now be attached through a series of connectorsthat vary in size, flexibility, or for circular diversity elements, thesize of the macrocycle. A further variation on this theme would be toregenerate not only the original diversity elements, but minorvariations (for example vary just one amino acid residue at a time froma cognate sequence) as well, to be combined with the diverse set ofconnector elements. This refined set of coferons would be rescreened inthe presence of the fluorescently labeled target protein at theappropriate concentrations to allow for selection of the tightestbinding combinations. The same principles of dynamic combinatorialchemistry described above would apply. The winning triplet of coferonswould be identified by their bar-code, DNA sequence tags, or by massspectroscopy. The coferon trimers selected by this protocol should haveaffinities to the target in the nanomolar to picomolar range.

A second version of the screen (similar to that in FIG. 11) depends onsome prior knowledge of potential ligands, or molecular modeling toidentify a set of potential binding elements. For example, the targetmay be a protein with a known binding pocket, such as a tyrosine kinase.Here, molecules known to or inferred to fit within the binding pocket(and chemical variants whose structure would occupy a similar3-dimensional space) are attached to a series of connectors that vary insize and flexibility. It is assumed (but should also be experimentallyverified) that the majority of members of this library would bind to thetarget with micromolar or even nanomolar affinities. Subsequently, thisfirst library of binding pocket ligands is combined with a secondlibrary of coferons with various diversity elements on beads, and athird library of diversity elements in solution. The trimers of bindingelements are screened as described above. The coferon trimers selectedby this protocol should inhibit enzymatic function if the binding pocketalso has an active site, and have affinities to the target in thenanomolar to picomolar range.

Screening for A-B-A-B Target Binding Coferons

Three types of geometries for these tetrameric coferons are envisioned.The first alternates the A and B linker elements. The simplest versionof such linker elements would contain aromatic rings that stack on eachother. The second geometry has an A-A dimer binding to a B-B dimer. Thismay be achieved using linker elements that form head-to-head homodimers,but also have groups that allow for linking of the two dimers on theirsides to achieve bonding between the four linker element groups. Thedimers may not stack directly over one another, but may be offset. Thethird geometry has an A-B dimer binding to a B-A dimer. This may beachieved using linker elements that form head-to-head heterodimers, butalso have groups that allow for linking of the two dimers on their sidesto achieve 90° C. angles between the four linker element groups. Forexample, linker elements based on nucleotide analogue base hydrogenbonding or base intercalation could form A-B heterodimers, and two A-Bheterodimers could stack on each other by taking advantage of aromaticstacking interactions, held in place by additional covalent bonding.

There are many variations for screening A-B-A-B tetramer coferons, whichfollow the initial variations for screening coferon dimers (FIGS. 8-12),except instead of synthesizing monomer diversity elements, onesynthesizes duplicate diversity elements and tethers the linker elementsto each other. Also, one may synthesize the “A” set of coferons on beadsor particles at a density that allows two such coferons to participatein forming an A-B-A-B tetramer, and then add the “B” coferons insolution.

Screening for Circular Sets of Target Binding Coferons

Two types of screens and two types of geometries for circular sets ofcoferons, ranging from tri-, tetra-, penta-, hexa-hepta- toocta-coferons, are envisioned. In the first design, all the coferons areidentical, i.e. circular A-A-A-A-A pentamer coferon. In the seconddesign, the coferons alternate, i.e. circular A-B-A-B-A-B hexamercoferon.

In designing screens for multiple coferons, it is important that thecoferons assemble in the proper order and correct number. One approachto achieving this is to create a circular scaffold onto which thecoferons or just the diversity elements are attached. Once the best setis identified, the coferons are resynthesized without the scaffold.

For example, a circular peptide of the form-Lys-Asp-Gly-Asp-Gly-Asp-Gly-Asp-Gly-Asp-Gly-Asp-(SEQ ID NO: 1) wouldallow for attachment to a bar-coded bead through the lysine gamma aminogroup. Ethylene glycol spacers of suitable length could then be attachedto all 6 aspartic acids. This would be followed by synthesis ofidentical diversity elements at all 6 positions. Such a “crown” designwould give the diversity elements the flexibility needed to bind to thetarget. Other crown type scaffolds include calixarenes andcyclodextrins.

An alternative variation of the above would attach linker elements thathave been optimized to assemble into hexagonal structures. The diversityelements could then be built directly from the linker elements, or toadd even more flexibility, use an additional ethylene glycol spacerbetween the linker elements and the diversity elements. This design isideally suited for identifying hexameric coferons composed of 6identical diversity elements.

To generate mixed diversity element coferons, i.e. a circularA-B-A-B-A-B hexamer coferon, one approach would be to start with acircular peptide of the form-Lys-Asp-Gly-Cys-Gly-Asp-Gly-Cys-Gly-Asp-Gly-Cys-(SEQ ID NO: 2). Again,the peptide is attached to a bar-coded bead through the lysine gammaamino group. Either the Cys or Asp groups may be protected to avoidfurther chemistry, ethylene glycol spacers attached to the remainingthree groups, then linker elements, and then identical diversityelements are built on those 3 chains. Once the first three diversityelements are built, the protecting groups on the other three amino acidsare removed, and the process repeated by adding ethylene glycol spacersand linker elements to the remaining three groups. Since the beadsalready code for the “A” diversity elements, the “B” elements are nowsynthesized as identical “cores” on every bead, for example usingalanine for every position. Alternatively, if there are an excess numberof beads containing the identical “A” diversity elements, limiteddiversity could be tested for the “B” coferon position, by dividing thebeads into 3N wells, and synthesizing the “B” coferon with N different Rgroups in the first diversity position, or N different R groups in thesecond diversity position, or N different R groups in the thirddiversity position. A given well will have a large number (i.e.1,000,000) beads containing a large diversity of structures in the “A”coferon position, but a given bead will have the identical “A” diversityin all three coferons that were synthesized off the circular peptide.All the beads in this single well will have the identical “B” coferon inall three positions. Fluorescently labeled target protein is added tothe beads (containing diverse “A” coferon and identical “B” coferons),and after a suitable period of panning, wells containing fluorescentlylabeled beads are identified. If the beads are in a single chamber, thebar-code is identified for the fluorescently labeled beads.

Once the best candidate “A” coferons have been identified (with orwithout the added limited diversity of the “B” coferons) the process isrepeated, however this time the beads contain molecules that arediversified in the “B” positions, while the “A” positions are limited tothe best “A” coferon(s). (Again, if there are an excess number of beadscontaining the identical “B” diversity elements, limited diversity couldbe included for the “A” coferon). Fluorescently labeled target proteinis added to the beads (containing diverse “B” coferon and identical “A”coferons), and after a suitable period of panning, wells containingfluorescently labeled beads are identified. If the beads are in a singlechamber, the bar-code is identified for the fluorescently labeled beads.This process first optimized the “A” coferon, and then the “B” coferon,and may be reiterated if needed, to find the best candidate diversityelements. The coferon hexamers (comprised of 3 A-B dimers) selected bythis protocol should have affinities to the target in the nanomolar topicomolar range.

Another approach relies on synthesis of the “A” and “B” set coferons onseparate macromolecules, allowing for screening diversity in bothcoferons simultaneously. For example, the “A” coferons could be attachedthrough ethylene glycol spacers to the 4 meso positions of a porphyrinring. Likewise, the “B” coferons could be attached through ethyleneglycol spacers to the 4 meso positions of a second porphyrin ring. Thetwo structures could interact through aromatic stacking of the twoporphyrin rings. If the rings are coaxial, the diversity elements couldbe in an alternating geometry, allowing for all 8 coferon diversityelements to fall to one side and bind the target with high affinity. Onecoferon set could be on a bead, with the other set in solution, allowingfor screening of both sets simultaneously.

In all the above cases, once the winning diversity element “multiplex”has been identified, the coferons are re-synthesized with monomerdiversity elements with the suitable linkers to allow for multimercoferon binding to suitable targets.

Selection Based on Screening

Coferons may be thought of as miniature antibodies that may disassembleoutside a cell and reassemble inside a cell to influence macromoleculeinteractions. There are two issues at play, how well the coferon candistinguish between the correct target and other closely related targets(i.e. specificity), and how it modulates the biological activity inquestion.

The evolutionarily driven selections described above are all based onbinding to the target, but they do not address binding to a specificsurface or face of the target, nor do they address the specificityissue. For example, aptamers can be selected for to bind known proteinswith very high binding affinities; however, these often turn out to bedriven by the negatively charged DNA backbone interacting withpositively charged residues on the protein target—and such aptamersoften have substantial non-specific binding to incorrect targets.

With current recombinant techniques, it is straightforward to generatepurified wild-type and specific mutant variants of virtually anyprotein, covalently attach protein targets to solid surfaces such asbeads, as well as fluorescently label such proteins. In addition, thereare several reagents for attaching fluorescent and quenching groups ontosmall molecules, binding ligands etc. Combinations of such groups may beused to detect close binding of two macromolecules by observing a FRETsignal, or conversely, detect two macromolecules no longer binding byseparating the fluorescent group from a nearby quenching group. Finally,for many protein targets that require an energy source, such as ATP, tosignal or function properly, there are many analogues which may “freeze”the protein in either an “active” or “inactive” conformation.

Selecting coferons to bind to a particular face or substrate-bindingpocket of a protein. Under these conditions a non-binding target proteinis synthesized or engineered, wherein the protein contains one or moremutations or chemical modifications or inhibitor binding to the face inquestion, such that the non-binding target protein no longer has theability to bind its partner protein, or substrate.

When one coferon is attached to a bead, and the binding of protein isdetected using a fluorescently labeled protein: Add unlabeled engineerednon-binding target protein at a molar excess to the labeled targetprotein, for example at a 100:1 excess. Beads containing coferon pairsthat bind uniquely to the target protein but not the engineerednon-binding target protein will bind fluorescently labeled protein andcan then be distinguished.

When the protein is attached to beads, and the coferon selected bytighter binding to the protein on beads: target proteins can be attachedto magnetic beads, or coded beads that may be separated from the otherbeads. Engineered non-binding target protein may be attached to otherbeads, which are present at a greater level, for example at a 100:1excess. Excess beads containing engineered non-binding target proteinwill swamp out coferons binding at the wrong surface. However, coferonsbinding the correct surface of target proteins may be selected by (i)magnetic separation or (ii) FACS sorting of these beads, respectively.

Selecting coferons to bind to a particular conformation of the protein,for example when it is binding ATP. Under these conditions, anon-reversible ATP analogue is used to bind to the protein to “freeze”it in the active conformation. Under these conditions a non-analoguebinding target protein is synthesized or engineered, where the proteincontains one or more mutations or chemical modifications, such that thenon-analogue binding target protein no longer has the ability to“freeze” it in the active conformation.

When one coferon is attached to a bead, and the binding of protein inthe active conformation is detected using a fluorescently labeledprotein bound to the non-reversible analogue substrate, unlabeledengineered non-analogue binding target protein is added at a molarexcess to the labeled target protein, for example at a 100:1 excess.Beads containing coferon pairs that bind uniquely to the target proteinbut not the engineered non-analogue binding target protein will bind tofluorescently labeled protein and can then be distinguished.

When the protein in the active conformation is attached to beads, andthe coferon selected by tighter binding to the protein on beads, targetproteins in the active conformation are attached (by using thenon-reversible analogue substrate) to magnetic beads, or coded beadsthat may be separated from the other beads. Engineered non-analoguebinding target protein are attached to other beads, which are present ata greater level, for example at a 100:1 excess. Excess beads containingengineered non-analogue binding target protein will inhibit coferonsbinding the wrong conformation. However, coferons binding the correctconformation of target proteins may be selected by (i) magneticseparation or (ii) FACS sorting of these beads, respectively.

Coferons can be selected to bind to a particular face of a protein tointerfere with that protein binding a second protein.

When one coferon is attached to a bead, and the binding of targetprotein is detected using a fluorescently labeled protein, a targetprotein with a fluorescent signal, and an excess of secondary proteinwith quenching group(s) that binds to the target protein are used toquench the fluorescent signal. Beads containing coferon pairs that binduniquely to the target protein in a way that interferes with binding ofthe second protein will bind fluorescently labeled protein and can thenbe distinguished.

Coferons can be selected to bind to enhance a protein-protein bindinginteraction.

When one coferon is attached to a bead, and the binding of targetprotein is detected using a fluorescently labeled protein, use a targetprotein with a fluorescent signal, and a secondary protein with anotherfluorescent group that will generate a FRET signal when binding to thetarget protein. Beads containing coferon pairs that bind uniquely to thetarget protein and second target protein so as to enhance theirinteraction will generate a FRET signal and can then be distinguished.

Coferons can be selected to inhibit or enhance enzymatic action orprotein function.

When one coferon is attached to a bead, and the binding of targetprotein is detected using a fluorescently labeled protein, those beadswhich are fluorescently labeled are selected, indicating binding ofproteins into microtiter wells, and assay for individual proteinactivity.

Therapeutics

An additional embodiment of the present invention relates to atherapeutic multimer which includes a plurality of covalently ornon-covalently linked monomers. Each monomer comprises a diversityelement which binds to a target molecule with a dissociation constant ofless than 300 μM and a linker element having a molecular weight lessthan 500 daltons, and capable of forming a reversible covalent bond ornon-covalent tight interaction with a binding partner of the linkerelement with a dissociation constant less than 30 μM, with or without aco-factor under physiological conditions. The diversity element and thelinker element are joined together for each monomer. The plurality ofmonomers are covalently bonded together or non-covalently linkedtogether through their linker elements, and the diversity elements forthe plurality of monomers bind to proximate locations of the targetmolecule.

A method of treating a subject for a condition associated with targetmolecule can be carried out by providing the therapeutic dimer,selecting a subject with the condition, and administering the treatmentdimer to the selected subject under conditions effective to treat thecondition.

Another embodiment of the present invention relates to a plurality oftherapeutic monomers capable of combining to form a therapeuticmultimer. Each monomer includes a diversity element which binds to atarget molecule and a linker element having a molecular weight less than500 daltons and capable of forming a covalent bond or non-covalent tightinteraction with a binding partner of the linker element with adissociation constant less than 300 μM, with or without a co-factor,under physiological conditions. The diversity element, which has adissociation constant less than 300 μM, and the linker element areconnected together directly or indirectly through a connector for eachmonomer. A plurality of monomers are capable of covalently bondingtogether or being non-covalently linked together through their linkerelements, and the diversity elements for the plurality of monomers bindto proximate locations of the target molecule.

A method of treating a subject for a condition associated with targetmolecule is carried out by providing a plurality of the therapeuticmonomers, selecting a subject with the condition, administering theplurality of treatment monomers to the selected subject under conditionseffective to treat the condition.

Therapeutic dimers are those dimers from which DNA and beads have beenremoved. These are shown in FIGS. 2.1E, where there is a connector, andFIG. 2J where there is no connector.

Therapeutically effective doses of compounds of the present inventionmay be administered orally, topically, parenterally, by inhalationspray, or rectally in dosage unit formulations containing conventionalnon-toxic pharmaceutically acceptable carriers, adjuvants, and vehicles.The term parenteral, as used herein, includes subcutaneous injections,intravenous, intramuscular, intrasternal injection, or infusiontechniques.

The pharmaceutical compositions containing the active ingredient may bein the form suitable for oral use, for example, as tablets, troches,lozenges, aqueous or oily suspensions, dispersible powders or granules,emulsions, hard or soft capsules, or syrups or elixirs. Thepharmaceutical compositions of the present invention contain the activeingredient formulated with one or more pharmaceutical excipients. Asused herein, the term “pharmaceutical excipient” means a non-toxic,inert solid, semi-solid or liquid filler, diluent, encapsulatingmaterial, or formulation auxiliary of any type. Some examples ofpharmaceutical excipients are sugars such as lactose, glucose, andsucrose; starches such as corn starch or potato starch; cellulose andits derivatives such as sodium carboxymethyl cellulose, ethyl cellulose,and cellulose acetate; powdered tragacanth; malt; gelatin; talc;excipients such as cocoa butter and suppository waxes; oils such aspeanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, cornoil, and soybean oil; glycols such as propylene glycol; esters such asethyl oleate and ethyl laurate; agar; buffering agents such as magnesiumhydroxide and aluminum hydroxide; alginic acid; pyrogen-free water;isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffersolutions; non-toxic, compatible lubricants such as sodium laurylsulfate and magnesium stearate; as well as coloring agents, releasingagents, sweetening, and flavoring and perfuming agents. Preservativesand antioxidants, such as ethyl or n-propyl p-hydroxybenzoate, can alsobe included in the pharmaceutical compositions.

Dosage forms for topical or transdermal administration of compoundsdisclosed in the present invention include ointments, pastes, creams,lotions, gels, plasters, cataplasms, powders, solutions, sprays,inhalants, or patches. The active component is admixed under sterileconditions with a pharmaceutically acceptable carrier and any neededpreservatives or buffers, as may be required. The ointments, pastes,creams and gels may contain, in addition to an active compound of thepresent invention, excipients such as animal and vegetable fats, oils,waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc and zincoxide, or mixtures thereof.

For nasal administration, compounds disclosed in the present inventioncan be administered, as suitable, in liquid or powdered form from anasal applicator. Forms suitable for ophthalmic use will includelotions, tinctures, gels, ointment and ophthalmic inserts, as known inthe art. For rectal administration (topical therapy of the colon),compounds of the present invention may be administered in suppository orenema form, in solution in particular, for example in vegetable oil orin an oily system for use as a retention enema.

Compounds disclosed in the present invention may be delivered to thelungs by the inhaled route either in nebulizer form or as a dry powder.The advantage of the inhaled route, over the systemic route, in thetreatment of asthma and other diseases of airflow obstruction and/orchronic sinusitis, is that patients are exposed to very small quantitiesof the drug and the compound is delivered directly to the site ofaction.

Dosages of compounds of the present invention employed will varydepending on the site of treatment, the particular condition to betreated, the severity of the condition, the subject to be treated (whomay vary in body weight, age, general health, sex, and other factors) aswell as the effect desired.

The amount of active ingredient that may be combined with thepharmaceutical carrier materials to produce a single dosage form willvary depending upon the host treated and the particular mode ofadministration.

The target molecule can be selected from the group consisting of: (1)G-protein coupled receptors; (2) nuclear receptors; (3) voltage gatedion channels; (4) ligand gated ion channels; (5) receptor tyrosinekinases; (6) growth factors; (7) proteases; (8) sequence specificproteases; (9) phosphatases; (10) protein kinases; (11) bioactivelipids; (12) cytokines; (13) chemokines; (14) ubiquitin ligases; (15)viral regulators; (16) cell division proteins; (17) scaffold proteins;(18) DNA repair proteins; (19) bacterial ribosomes; (20) histonedeacetylases; (21) apoptosis regulators; (22) chaperone proteins; (23)serine/threonine protein kinases; (24) cyclin dependent kinases; (25)growth factor receptors; (26) proteasome; (27) signaling proteincomplexes; (28) protein/nucleic acid transporters; and (29) viralcapsids.

The therapeutic multimer, or plurality of therapeutic monomers containsone or more known ligands as diversity elements and achieves greaterefficacy against both wild-type and mutant variants of the targetmolecule than would be achieved with a single ligand.

The therapeutic multimer or plurality of therapeutic monomers bind to ormimics one or more of the domains selected from the group consisting ofSH2, FHA, 14-3-3, WW, WD40, MH2, BROMO, UBA, PTB, SH3, EVH1, GYF, VHS,PDZ, PUF, TUBBY, SAM, DD, CARD, PyD, PB1, BRCT, PH, FYVE, C1, FERM, C2,PX, and ENTH.

The therapeutic multimer or plurality of monomers either interfereswith, inhibits binding of, or inhibits activation of the following: (1)target cleavage of a substrate, by binding to the target with adissociation constant that is less than or equal to the dissociationconstant of the substrate from the target; (2) binding of a bindingprotein to a target, by binding to the target with a dissociationconstant that is less than or equal to the dissociation constant of thebinding protein; (3) inactivation of a target that by a binding partner,by binding to the target and mimicking the binding partner; (4)inactivation of a target or mutant target by a binding partner, bybinding to an inactivating binding partner-target complex orinactivating binding partner-mutant target complex; (5) binding of afirst binding partner to a target, by binding to the target andrecruiting a second binding partner to bind to the target and themultimer and forming a multimer-target-second binding protein complex,whose dissociation constant is less than or equal to the dissociationconstant of the first binding protein; (6) binding to a receptor target,by binding to the receptor target and interfering with receptordimerization; (7) binding to a binding partner by reducing itsrecruitment to a receptor target, by binding the receptor target at aligand binding site to act as an antagonist, or binding the receptortarget at the binding partner binding site to act as an antagonist; (8)polymerization of a target into filaments, by binding on a monomer ordimer target; and (13) aggregation of a target, by binding a monomer ordimer target.

The therapeutic multimer or plurality of therapeutic monomers eitherenhances activation of, enhances binding of, or activates the following:(1) activation of a target by a binding partner, by binding to thetarget and mimicking the binding partner; (2) activation of a target ormutant target by a binding partner, by binding to an activating bindingpartner-target complex or activating binding partner-mutant targetcomplex; (3) a first weak binding partner to a target, by binding to thetarget and recruiting a second binding partner to bind to the target,multimer, and first binding partner and forming a multimer-target-secondbinding protein complex, or forming a multimer-target-first bindingprotein-second binding protein complex; (4) a receptor target by bindingto the receptor target at the ligand binding site, and facilitatingreceptor dimerization; (5) a receptor target by binding to an allostericsite on the receptor target and facilitating receptor dimerization inthe presence of activating ligand; and (6) a binding partner that isrecruited to a receptor target by a ligand binding to the receptortarget, by binding to the receptor target at the ligand binding site toact as an agonist, which recruits and activates the binding partner, orbinding to the receptor target and the ligand or the receptor target andthe binding partner, to accelerate recruitment and activation of thebinding partner.

The therapeutic multimer or plurality of therapeutic monomers eitherenhances or alters protein metabolism by: (1) stabilizing target ormutant target folding; (2) enhancing or interfering with a covalentsignaling event; (3) mimicking a covalent signaling event; (4)inhibiting multi-subunit assembly; (5) inhibiting multi-subunitdisassembly; or (6) inhibiting degradation by binding the target ortarget binding partner.

The therapeutic multimer or plurality of therapeutic monomers interfereswith, activates, enhances, or mimics covalent modification of the targetby phosphorylation, dephosphorylation, acetylation, methylation,sumolation, ubiquitination, farnesylation, and addition of sugar andcarbohydrate moieties, by binding to the target or the target-modifyingenzyme complex to inhibit, activate, enhance, or modulate proteinsignaling, transport, or degradation through additional proteininteractions.

The therapeutic multimer or plurality of therapeutic monomers interfereswith or inhibits either: (1) an essential viral target from a set oftargets that includes reverse transcriptase, protease, or viralintegration proteins, by providing a plurality of monomers that can bindat a first site, and a plurality of monomers that can bind at anadjacent second site, said plurality of monomers creating a cocktail oftherapeutic multimers providing broad inhibition of viral target andmutant variant viral targets; (2) viral entry into cells by binding toand inhibiting the cellular receptor responsible for assisting viralentry; (3) a cellular protein that assists with viral function; or (4) aviral protein such that it no longer inhibits a host defense protein.

The therapeutic multimer has a dissociation constant from the targetmolecule that is from within a ten-fold range of about 47 pM or lower towithin a ten-fold range of 48 nM such that binding of the therapeuticmultimer to the target molecule is sufficient to displace anotherprotein, protein domain, macromolecule, or substrate with an equal orhigher dissociation constant from binding to the target protein, or isof sufficiently tight binding to activate, enhance, or inhibit thebiological activity of the target molecule or its binding partners suchthat about 70% to 100% of the target molecule within the target cells isbound by the therapeutic multimer to achieve the desired therapeuticeffect. This method includes providing a first monomer, wherein thedissociation constant of the diversity element from the target moleculeis from within a three-fold range of 100 nM to within a three-fold rangeof 10 μM. A second monomer, wherein the dissociation constant of thediversity element from the target molecule is from within a three-foldrange of 1 μM to within a three-fold range of 10 μM is also provided.The dissociation constant between the linker element of the firstmonomer and its binding partner of the second monomer is from within athree-fold range of 10 μM to within a three-fold range of 100 μM. Theconnector joining the linker element to the diversity element for eachmonomer is in the range of about 2 or less rotatable bonds to about 5rotatable bonds. The therapeutic multimer is present so that thesteady-state concentrations of the monomers in the blood are in therange of from about 0.1 μM to about 5.0 μM or higher.

PROPHETIC EXAMPLES

The following prophetic examples sets forth the procedure forpreparation of coferons. The steps include: 1) synthesis of bead-boundand solution libraries of linker element monomers; 2) screening andselection of the pair of linker element monomers that provide thetightest binding linker element dimer; 3) synthesis of a library ofdiversity elements attached to each of the selected linker elementmonomers to generate a library of coferon monomers; and 4) screening andselection of the coferon dimers that have the highest affinity for atarget protein.

Prophetic Example 1 Synthesis of a Library of Linker Element Monomers

In this portion of the prophetic example, as described in FIG. 30, thesynthesis of linker elements of the type Linker Element 9, is shown bythe generic Formula XIII, an aromatic compound that intercalates withone or more of its binding partners, is described. For the processesbelow, beads are used where each bead has a unique identifier. Thisidentifier may be a Veracode™ barcode, a sequence of DNA, a set ofmolecules that may be distinguished spectrally, or a set of moleculesthat may be distinguished by their mass. In general, the number of beadsexceeds the number of different final product structures, such that morethan one barcode can encode for the same final product on independentbeads, but no two beads have the same barcode and different productmolecules (other than stereoisomers). Ten different sets of Veracode™beads (each bead bearing a different barcode) are individually reactedwith 10 different alkynes to form a covalent bond between the alkynesand the beads. This produces a set of alkynes of the formula (1)covalently attached to beads where Ar₁ through Ar₁₀ represent 10different aromatic groups. The alkyne added to a given individual beadis determined by reading the barcodes of each bead in each of the 10sets, either before or after the alkyne reaction. The alkynes may beattached to Veracode™ beads functionalized with —NH₂, —COOH, or otherreactive groups, such that the covalent bond formed between the aromaticalkynes and the beads may be cleaved with the use of appropriatereagents. The beads are then pooled, split into 10 new different sets,and each set reacted with one of 10 different azides of the formula (2)where Ar₁₁ through Ar₂₀ represent 10 different aromatic groups. Theazide added to a given individual bead is determined by reading thebarcodes of each bead in each of the 10 sets, either before or after theazide reaction. The reaction results in the synthesis of a library of100 distinct disubstituted 1,2,3-triazoles of the formula (3) covalentlyattached to the Veracode™ beads. The nature of the particular aromaticrings attached to the triazole on a given bead may be deduced from thebarcode encoding each bead. The beads bearing the disubstituted1,2,3-triazoles are then reacted with cysteine to form a library of beadbound disubstituted 1,2,3-triazole linker elements of the formula (4),which bears a cysteine residue. The cysteine residue may be added in itsdisulfide form to prevent the cysteine thiol from competing with theamination. In this case, subsequent treatment with thioethanol willgenerate the free cysteine thiol.

The process is then repeated as shown in FIG. 31, where 10 new sets ofdifferent Veracode™ beads (each bead bearing a different barcode) areindividually reacted with 10 different azides. This produces a set ofazides of the formula (5) covalently attached to beads where Ar₁ throughAr₁₀ represent 10 different aromatic groups. The azide added to a givenindividual bead is determined by reading the barcodes of each bead ineach of the 10 sets, either before or after the azide reaction. Thebeads are then pooled, split into 10 new different sets, and each setreacted with one of 10 different alkynes of the formula (6) where Ar₁₁through Ar₂₀ represent 10 different aromatic groups. The alkyne added toa given individual bead is determined by reading the barcodes of eachbead in each of the 10 sets, either before or after the alkyne reaction.The reaction results in the synthesis of a library of 100 distinctdisubstituted 1,2,3-triazoles of the formula (7) covalently attached tothe Veracode™ beads. The beads bearing the disubstituted 1,2,3-triazolesare then reacted with cysteine to form a library of bead bounddisubstituted 1,2,3-triazole linker elements of the formula (8), whichbears a cysteine residue. The cysteine residue may be added in itsdisulfide form to prevent the cysteine thiol from competing with theamination. In this case, subsequent treatment with thioethanol willgenerate the free cysteine thiol. This is the Linker Element Library 2.

The beads comprising the two Linker Element Libraries (Library 1 and 2)are first combined and then split into equal halves. One half of thebeads are retained as the bead set of linker element monomers. This setis reacted with beta-mercaptoethanol, or a similar agent to create amixed disulfide link. The other half of the beads is treated with theappropriate reagents to cleave the linker elements from the beads andrelease them into the solution. The released linker element moleculesare then reacted with a fluorescent dye such that they are covalentlylinked to the fluorescent dye. These fluorescently labeled linkerelement monomers are the solution set of linker element monomers. SeeFIG. 32.

Prophetic Example 2 Screening of Linker Element Monomers to Find Pairsof Linker Elements that Bind Each Other to With the Greatest Affinity

The bead set of linker element monomers is allowed to interact with thesolution set of linker element monomers in an aqueous buffer that mimicsphysiological conditions (pH 7.4, 37° C.). The linker elements insolution intercalate with the linker elements on beads in a reversiblemanner. For the tightest binding pair of linker element monomers, thisequilibrium is shifted towards the formation of linker element dimers(FIG. 33). In this portion of the prophetic example, the greatestaffinity is seen between the monomer containing the aromatic groups Ar₁and Ar₂ and the monomer containing the aromatic groups Ar₃ and Ar₄. Thelinker element monomer 1,2,3-triazole flanked by the aromatic groups Ar₁and Ar₂, is bound to a Veracode™ bead and can be identified based on thebarcode on the Veracode™ bead. The linker element monomer 1,2,3-triazoleflanked by the aromatic groups Ar₃ and Ar₄, is in solution and alsobears a fluorescent label. When the solution is screened, beads thatprovide a fluorescent signal are selected. The Veracode™ barcodeidentifies the linker element monomer attached to the bead (with Ar₁ andAr₂). The solution also contains the exact same linker element monomerswhere the monomer containing the aromatic groups Ar₁ and Ar₂ is insolution and bears a fluorescent label and the monomer containing thearomatic groups Ar₃ and Ar₄ is attached to a Veracode™ bead with adifferent barcode. The dimers formed between these linker elementmonomers are also selected and the Veracode™ barcode identifies thelinker element monomer attached to the bead (with Ar₃ and Ar₄).

In the next step, diversity element libraries are synthesized on each ofthese linker element monomers.

Prophetic Example 3 Synthesis of a Library of Diversity ElementsAttached to Each of the Selected Linker Element Monomers to Generate aLibrary of Coferon Monomers

A diversity element library based on a tri-substituted cyclopentanescaffold is synthesized and attached to the first linker element. Thelinker element is re-synthesized on a set of Veracode™ beads, each beadcontaining a unique barcode (formula (9)) and reacted with 10 differentamino acids (formula (10)) to form a library of linker element-aminoacids of the formula (11). The Veracode™ barcode identifies theparticular amino acid attached to the linker element. The side chains ofthe amino acids represent an area of diversity in the final coferonmonomer. The bead bound library is then reacted with thionyl chloride toconvert the carboxylic acid groups of the amino acids to acid chloridesthus forming a bead bound library of linker element-amino acid chlorides(formula 12)). See FIG. 34.

As shown in FIG. 35, (1R,3R,4S)-3,4-diaminocyclopentanol (13) is reactedwith a resin to covalently link one of the amino groups to the resin.The resin bound structure (14) is then treated withN,N′-Diisopropylcarbodiimide (DIC) and reacted in 10 different reactionvessels with one each of 10 different carboxylic acids R₁₋₁₀COOH whereR₁₋₁₀ represent 10 different aliphatic, alicyclic, or aromatic groups toform amide linkages between the free amine of the cylopentanol and theacid (15). These carboxylic acid amides represent another area ofdiversity in the final coferon monomer. Transiently formed estersbetween the carboxylic acids and the hydroxyl group of the cyclopentanolare hydrolyzed with a mixture of ammonia and methanol. Each individualset of amides (15) is treated with trifluoroacetic acid (TFA) andtriisopropylsilane (TIS) to cleave each set from the resin to generate10 different sets of cyclopentanol amides with a free amino group (16)and then reacted with monomethoxytrityl chloride (MMTrCl) to protect theamino group (17). The identity of each R group is known since the setsare kept separate.

As shown in FIG. 36, the 10 sets of cyclopentanol amides (17) are thenreacted with the bead bound library of linker element-amino acidchlorides (12) in 4-dimethylaminopyridine (DMAP) to form a library oflinker elements where the amino acids form an ester linkage with thehydroxyl group of the cyclopentanol amides (18). The carboxylic acidadded to a given individual bead is determined by reading the barcodesof each bead in each of the 10 sets, either before or after the amidereaction. The molecules are then treated with trifluoroacetic acid (TFA)in dichloromethane (DCM) to remove the monomethoxytrityl protectinggroup to generate the free amine (19).

The beads are pooled to generate a library, which now contains beadsattached to linker element monomers that bear a cyclopentane scaffoldwith 10 different esters at position 1, 10 different amides at position3 and a free amino group at position 4 (19). The library is then splitinto 10 sets, and each set is treated with one of 10 differentcarboxylic acids R₁₁₋₂₀COOH where R₁₁₋₂₀ represent 10 differentaliphatic, alicyclic, or aromatic groups and dicyclohexyl carbodiimide(DCC) to form amides between the carboxylic acids and the amino group atposition 4 of the cyclopentane scaffold to form the library of coferonmonomers (20), as shown in FIG. 37. The carboxylic acid added to a givenindividual bead is determined by reading the barcodes of each bead ineach of the 10 sets, either before or after the amide reaction. Thesecarboxylic acid amides represent a third area of diversity in the finalcoferon monomer. FIG. 38 shows the three distinct areas of structuraldiversity in the coferon monomer overlaid with green, yellow and magentaovals.

The entire process is repeated with the second linker element monomer(to create the diversity element library attached to the linker elementwith Ar₃ and Ar₄. The library is released from the beads and used inconjunction with the bead bound library above (where the linker elementmonomer contains Ar₁ and Ar₂) to screen for coferon dimers with thegreatest affinity for a target protein.

Prophetic Example 4 Screening of Coferon Monomers to Find Pairs ofCoferons that Bind to a Target Protein with the Greatest Affinity

The target protein is labeled with a fluorophore and incubated with thetwo coferon libraries, one coferon library bound to Veracode™ beads andthe other coferon library in solution. The coferon monomers bind to thetarget reversibly. Coferon monomers that bind to the target with greateraffinity are progressively removed from the solution as the equilibriumshifts from free monomer in solution to monomer bound to the protein forthese higher affinity monomers. Coferon monomers with the greatestaffinity interact with each other when bound to the protein and formcoferon dimers. Some coferon dimers comprise monomers where bothmonomers are attached to Veracode™ beads. Other coferon dimers comprisemonomers where both monomers are from the coferon library in solution. Athird set of coferon dimers comprise of monomers where one monomer isattached to a Veracode™ bead and the other monomer is in solution (SeeFIG. 39). Judicious choice of coferon linker elements may favor theformation of heterodimer coferons such that the majority of coferondimers contain one monomer on a Veracode™ bead and the other monomerfrom solution, both binding the target protein to the bead. After asuitable period of incubation, the beads are sorted to select for beadsthat provide the strongest fluorescent signal. The Veracode™ barcode onthe bead identifies one of the coferon monomers that binds to the targetprotein. To determine the identity of the second coferon monomer fromthe solution library, one repeats the experiment so that the librarieson the bead and in solution are reversed.

The coferon monomer pairs that make up the winning coferon dimers canthen be individually synthesized for use as therapeutic monomers.

These prophetic examples use 3 synthetic steps where each stepintroduces a 10-fold increase in diversity. The diversity is actuallyhigher due to stereoisomers and chirality in the various structures. Ata minimum, 1,000 diversity elements are generated for each coferonmonomer set. Thus, when used as a dimer, the diversity is 10⁶.

The above prophetic examples need not be limited to just 10 additionsper synthetic step. For example there are some 2,000 commerciallyavailable low molecular weight amino acids, and an equivalent order ofmagnitude of commercially available low molecular weight carboxylicacids.

In molecular biology, those skilled in the art of robotic instrumentsknow it is straightforward to perform reactions in a 96 well or 384 wellformat. For the purposes of the discussions below, these numbers can berounded up to 100 reactions and 400 reactions, to simplify thecalculations of diversity generated by the process. By way ofcomparison, the peptide recognition arms of individual antibodies have adiversity of the order of 10⁹ to 10¹².

When considering making a coferon monomer library comprised of threesteps with 100 diversity elements in each of the three steps, thediversity of the final library is 10⁶. If this library is then combinedwith another library in solution with an equal diversity, the dimerdiversity is now at 10¹², the same level as the best antibody diversityloop. However, it may turn out that the time to reach equilibriumthrough dynamic combinatorial chemistry is too long, or theconcentration of each reactant is too low. Consequently, background ornon-specific effects may impede identification of the winning coferonpair.

In order to overcome this possible limitation at high levels of coferondiversity, an alternative approach can be used for screening the coferondimers.

Since synthesis is straightforward in 96 well format (again, here itwill just be called 100 wells), the last step can keep these 100 wellsseparated as individual reaction chambers, both for those reactions thatare retained on beads, as well as those that are cleaved from the solidsupport. In such a case, one could screen 100 plates of 100 wells (inreality 96 plates of 96 wells), such that the individual diversity beingqueried in each well is only 10⁴×10⁴=10⁸, even though the overalldiversity being queried is 10⁶×10⁶=10¹².

Likewise, for synthesis in 384 well format (again, here it will just becalled 400 wells), the last step can keep these 400 wells separated asindividual reaction chambers, both for those reactions that are retainedon beads, as well as those that are cleaved from the solid support. Insuch a case, one could screen 400 plates of 400 wells (in reality 384plates of 384 wells), such that the individual diversity being queriedin each well is only 1.6×10⁵×1.6×10⁵=2.6×10¹⁰, even though the overalldiversity being queried is 6.4×10⁷×6.4×10⁷=4.1×10¹⁵. This level ofdiversity, allowing for easy screening and identification of the bestlead molecules is difficult to achieve by standard combinatorialchemistry means.

Prophetic Example 5 Simulation of Binding Equilibria for Coferon Dimerto Target Protein: Determination of Concentrations of Coferon DimersBinding to a Target Protein for Varying Concentrations of Each CoferonMonomer and Target Protein and for Varying Values of DissociationConstants for Each Coferon Monomer to the Target Protein and Between theCoferon Monomers

Most drugs function by binding to a biological target in competitionwith a natural substrate. In the case of infectious diseases, thiscompetitive binding can turn off an essential function and kill ordeactivate the infectious agent. In diseases such as cancer thecompetitive binding can either selectively kill malfunctioning cells orre-adjust endogenous biochemical processes so that cells can againfunction normally.

Two types of simulations of coferon dimers binding to target proteinshave been performed. In the first type of simulation, an excess ofcoferons over target (FIGS. 48-81) was used. In some of thesesimulations, the amount of coferons used would be larger than areasonable concentration in the blood stream. However, rarely is atarget present at high concentration throughout the entire body. Oftenthe cells that are being targeted by the drug have a higherconcentration of target protein than cells not targeted by drug. This isespecially true when the targeted cells are tumor cells.

A large tumor of half a pound (8 oz) represents only 1/300th to 1/400thof the average male body weight, and thus the vast majority of cellswill have far less target. Therefore, starting with even a lowconcentration of the coferons in the body will be sufficient to bind toand inhibit the protein target, even if the concentration of thatprotein target in the specific cells is 10 to 40-fold higher than in thebody.

When coferon monomers enter the target cell, they bind to the targetprotein. When they form dimers on the protein target, the concentrationsof monomers inside the cell decrease. To more accurately simulateconditions in vivo, the steady-state or “renewable” concentrations ofthe coferons were set at a given level, reflecting the ability toprovide a steady dose of drug over time (FIGS. 47-106). This may beillustrated by the following example: consider a situation where theconcentration of coferon monomers is at 1 μM outside the cell, but thetarget is at 10 μM inside the cell. When each monomer has a low K_(d)value (for example, 1 μM) of dissociation from the target, and the twomonomers have a low K_(d) value (for example 10 μM) of dissociation fromeach other, then approximately 95% of the monomers inside the cell willform a dimer on the target—even though at this point, 90% of the targetis still free. However, when the target binds monomer and coferon dimersinside the cell, the effective concentration of free monomer within thecell would decrease to about 0.05 μM. Thus, if the patient continues totake pills to keep the concentration of monomer in the blood stream at 1μM, after taking 10 pills (simplifying assuming good bioavailability andno excretion) the patient should achieve on the order of from 95% of thetarget bound, even when the target is present at a concentration of 10μM. Again, as mentioned above, in most cases only a very small fractionof the body will have the target protein at high concentration, so inpractice, it will take fewer pills.

The large contact surfaces, (˜1,500-3,000 Å²) of a protein-proteininteractions are several fold larger than protein-small moleculeinteractions (˜300-1,000 Å²). Typically, the dissociation constant ofprotein-protein interactions are in the nanomolar to micromolar range,often similar to the molecules that also bind these proteins. Forexample, K_(d) values for various protein-protein interactions are asfollows: p53-Mdm2, 600 nM; RAS-RAF, 80 nM; β-catenin-pAPC, 10 nM;Grb7-SH2, 11 μM; NF2-MPP1, 3.7 nM; CDC25B-Cdk2-pTyP/CycA, 10 nM (Table3).

TABLE 3 Examples of Dissociation Constants for Known ProteinInteractions DISSOCIATION PROTEIN SUBSTRATE CONSTANT (K_(d)) CDC25BCdk2-pTpY/CycA 10 nM⁽¹⁾ Bcl-xL cytochrome c 120 nM⁽²⁾ Bcl-xL BAK peptide200 nM⁽³⁾ DnaK GrpE 30 nM⁽⁴⁾ GABP β GABP β 600 nM⁽⁵⁾ Grb7 SH2 11 μM⁽⁶⁾NF2 MPP1 3.7 nM⁽⁷⁾ HPV E2 E1 60 nM⁽⁸⁾ IL-2Rα IL2 60 nM^((9, 10)) p53MDM2 600 nM⁽¹¹⁾ p53 DNA 14 μM⁽¹²⁾ p53 HmtSSB 12.7 μM⁽¹³⁾ pAKT 14-3-3 22nM⁽¹⁴⁾ AKT 14-3-3 27 nM⁽¹⁴⁾ c-Jun/c-Jun (AP1) DNA 190 nM^((15, 16)) ERKMEK1 610 nM⁽¹⁷⁾ CDK2 ATP 227 nM⁽¹⁸⁾ CDK2 ADP 2.7 μM⁽¹⁸⁾ CDK2 Cyclin A 48nM⁽¹⁸⁾ RAS RAF 80 nM⁽¹⁹⁾ CaMKII Calmodulin 30 nM⁽²⁰⁾ β CATENIN ICAT 3.1nM⁽²¹⁾ β CATENIN APC 310 μM⁽²¹⁾ β CATENIN pAPC 10 nM⁽²¹⁾ EGFR EGF 2nM⁽²²⁾ ZipA FtsZ-derived peptide 21.6 μM⁽²³⁾ ASK1 ASK1 220 nM⁽²⁴⁾ MKK4JNK3 35 μM⁽²⁵⁾ MEK2 ERK2 9 μM⁽²⁶⁾ MEK1 ERK1 29 μM⁽²⁶⁾ SMAC/DIABLOSMAC/DIABLO 35 zM⁽²⁷⁾ SMAC/DIABLO XIAP 800 nM⁽²⁸⁾ Apaf-1 Peptoid 1a 57nM⁽²⁹⁾All of the following citations are hereby incorporated by reference intheir entirety.

-   1. Sohn, J., et al., Biochemistry, 46: 807-18 (2007).-   2. Yadaiah, M., et al., Biochim Biophys Acta, 1774: 370-9 (2007).-   3. Sattler, M., et al., Science, 275:983-6 (1997).-   4. Gelinas, A. D., et al., J Mol Biol, 339:447-58 (2004).-   5. Desrosiers, D. C., et al., J Mol Biol, 354:375-84 (2005).-   6. Porter, C. J., et al., Eur Biophys J, 34:454-60 (2005).-   7. Seo, P.-S., et al., Exp Biol Med, 234:255-62 (2009).-   8. Abbate, E. A., et al., Genes Dev, 18:1981-96 (2004).-   9. Arkin, M. R., et al., Proc Natl Acad Sci USA, 100: 1603-8 (2003).-   10. Braisted, A. C., et al., J Am Chem Soc, 125 3714-5 (2003).-   11. Kussie, P. H., et al., Science, 274:948-53 (1996).-   12. Joerger, A. C., et al., J Biol Chem, 280:16030-7 (2005).-   13. Wong, T. S., et al., Nucleic Acids Res, 37: 568-81 (2009).-   14. Sadik, G., et al., J Neurochem, 108: 33-43 (2009).-   15. Kobayashi, T., et al., Anal Biochem, 332:58 (2004).-   16. John, M., et al., Nucleic Acids Res, 24:4487-94 (1996).-   17. Horiuchi, K. Y., et al., Biochemistry, 37: 8879-85 (1998).-   18. Heitz, F., et al., Biochemistry, 36: 4995-5003 (1997).-   19. Kiel, C., et al., J Mol Biol, 340:1039-58 (2004).-   20. Brokx, R. D., et al., J Biol Chem, 276:14083-91 (2001).-   21. Choi, H. J., et al., J Biol Chem, 281:1027-38 (2006).-   22. Swindle, C. S., et al., J Cell Biol, 154:459-68 (2001).-   23. Mosyak, L., et al., Embo J, 19:3179-91 (2000).-   24. Bunkoczi, G., et al., Structure, 15: 1215 (2007).-   25. Bardwell, L., et al., Methods, 40:213 (2006).-   26. Bardwell, A. J., et al., J Biol Chem, 276: 10374-86 (2001).-   27. Goncalves, R. B., et al., Biochemistry, 47:3832-41 (2008).-   28. Kipp, R. A., et al., Biochemistry, 41: 7344-9 (2002).-   29. Malet, G., et al., Cell Death Differ, 13: 1523-32 (2006).

In contrast, independent protein domains interacting with peptides orproteins can demonstrate K_(d) values up to millimolar ranges,demonstrating the importance of the complete protein structure forbinding affinities in addition to the other environmental factors thatdetermine pharmacokinetic properties. For example K_(d) values for thefollowing domain-domain interactions are: SH2-phosphotyrosine residue inGrb2, 0.2-11 μM; FHA-phospho-threonine and phospho-tyrosine residues inKIF13B, 1-100 μM; MH2-phospho-serine residues in SMAD2, 240 nM;PTB-phospho-tyrosine residues, Asn-Pro-X-Tyr motifs in IRS-1, 160 nM-10μM; SH3-proline-rich peptides with consensus Pro-X-X-Pro (SEQ ID NO. 3),where X is an amino acid. In Grb2, 1-500 μM; FYVE-phosphatidylinositol3-phosphate, zinc in SARA, 50 nM-140 μM (Table 2).

In order for a coferon dimer or multimer to be an effective inhibitor ofa given protein-protein interaction, the highest dissociation constantof the coferon dimer from the protein target (usually K_(d3) or K_(d4),but also K_(d6) when using irreversible linker elements) needs to belower than or about equal to the dissociation constant of the nativeprotein binding partner from the protein target.

The simulations are idealized. All of the simulations assumenon-competitive binding. That is, the site bound by the coferon dimer iseither not bound by any natural ligand or the natural ligand binds farless tightly and/or is at much lower concentration. It is not necessaryto correct for binding of the coferon dimer to other biomolecules in thesystem. The concentrations assumed for coferon monomers C1 and C2 areafter loss due to binding to serum albumin, etc. In the simulations, thedissociation constants for the three different dissociation pathways areprovided (see below). The highest value will be the pathway ofdissociation. If this value is lower than the value between the proteintarget and another protein binding partner, the coferon dimers willdisplace the protein binding partner. Even if the dissociation value forthe coferon dimer is higher than for the natural protein bindingpartner, the coferons may still have an inhibitory effect if used at ahigher concentration.

An effective drug is one that binds to a target with high affinity, butdoes not have significant affinity for other proteins and biologicalmolecules in the body. Such a drug is said to be selective for itstarget. Binding is characterized by a change in free energy designatedby the symbol ΔG. The greater the change in free energy on binding, thehigher the affinity. A negative value for ΔG indicates a spontaneousprocess. When ΔG is positive binding is not favorable.

One can estimate the dissociation constants, K_(d), for monomer ordimer-target complexes using standard equilibria expressions:

$\begin{matrix}{K_{d} = \left. {\frac{\lbrack L\rbrack\lbrack R\rbrack}{\lbrack{LR}\rbrack}\mspace{14mu}{and}\mspace{14mu}{the}\mspace{14mu}{equilibrium}\mspace{14mu}{{expression}\mspace{14mu}\lbrack{LR}\rbrack}}\rightleftarrows{\lbrack L\rbrack + \lbrack R\rbrack} \right.} & \left\lbrack {{Equation}\mspace{14mu} 1} \right\rbrack\end{matrix}$where [L] is the concentration of drug or ligand, [R] is theconcentration of receptor (typically a protein), and [LR] is theconcentration of the complex. K_(d) is typically in units of moles/liter(M) and is equivalent to the concentration of drug at which theconcentration of protein and protein-drug complex are equal (K_(d)=[L]when [R]=[LR]).K_(d) is related to the free energy by the equations:ΔG=−RT ln K _(d)  [Equation 2]or K _(d) =e ^(−ΔG/RT)  [Equation 3]

At 37° C. ln K_(d)=−ΔG/(8.3145 J/K)(310 K) when ΔG is given in J/mol.

FIG. 40, is a schematic representation of a system of two coferonmonomers (C1 and C2) that bind to orthogonal sites on a protein target(T) and to each other. Coferon 1 is illustrated as a green shape,coferon 2 as a blue shape, and the target protein as a white shape. Theequilibrium of coferon 1 (C1) binding to the target is given by thedissociation constant K_(d1). The equilibrium of coferon 2 (C2) bindingto the target is given by the dissociation constant K_(d2). In turn,coferon C2 may bind the complex of C1T (with dissociation constant ofK_(d3)), while coferon C1 may bind the complex of C2T (with dissociationconstant of K_(d4)). The C1-C2 coferon dimer itself is in equilibriumwith the target, with dissociation constant of K_(d6). Finally, thedimer will dissociate to form the two monomers, with dissociationconstant of K_(d5).

The enhanced binding achieved by coferon dimers can be understood byconsideration of an ideal case it which each monomer does not compromisethe binding of the other monomer and the entropy loss of the tertiarycomplex is no greater than the sum of the entropy losses for each twocomponent binding step. If a ligand such as coferon monomer 1 binds to aprotein target with a gain in free energy equal to ΔG1, then the finalassembly step by addition of coferon monomer 2 would occur with a freeenergy change ΔG4. In the ideal case ΔG4=ΔG1+ΔG5, andK_(d)=e−(ΔG1+ΔG5)/RT. The assembled complex (PE FIG. 40) can dissociateby any of three pathways (A-C) with the dissociation free energies givenby:ΔG3=ΔG2+ΔG5 ln K _(d3)=−(ΔG2+ΔG5)/(8.3145 J/K) (310 K)  Path AΔG4=ΔG1+ΔG5 ln K _(d4)=−(ΔG1+ΔG5)/(8.3145 J/K) (310 K)  Path BΔG6=ΔG1+ΔG2 ln K _(d5)=−(ΔG1+ΔG2)/(8.3145 J/K) (310 K)  Path C

The preferred pathway for dissociation will be the one with the lowestvalue of ΔG dissociation.

The free energy of binding between the two coferon monomers, ΔG5 can betuned by varying the structure of the linker elements within each familyin multiple ways, including incorporation of electron withdrawing and/orreleasing groups, addition or subtraction of groups that influencesteric interactions, and the addition or subtraction of groups thatinfluence rotational freedom of bonds. The ability to adjust the K_(d)of linker elements allows one to vary the concentration of coferonmonomer commensurate with necessary therapeutic dose.

In many of the molecular designs, the linker elements bind to each othervia a covalent association, depicted as a red dot in FIG. 41. Thecovalent association between the linker elements may be eitherreversible, or essentially irreversible. One example of essentiallyirreversible linker elements is the formation of thiazolines by reactionof β-aminothiols (e.g. cysteine) with aldehydes or ketones. Formation ofa covalent bond between the monomers is facilitated by the targetprotein, because it creates a high local concentration of the twoindividual monomers at the target surface. Moreover the thiol componentwould be protected as a disulfide and not undergo conversion to the freethiol until the disulfide prodrug enters the cell and is cleaved byglutathione. Note that when both monomers bind to the target, the twolinker elements are brought in close proximity with each other,effectively increasing their relative concentration to 1M or higher. Ifformation of the covalent bond(s) is irreversible, the K_(d5)dissociation value will be zero. Under these conditions, dissociation ofthe coferon dimer from the target protein will be exclusively throughK_(d6), and may be 100-fold or even 1,000-fold lower.

In some cases, covalent linkage between two monomers may decrease thebinding contributions of each monomer. In these cases, ΔG3 may be lessthan the sum ΔG1+ΔG5 and/or ΔG4 less than the sum ΔG2+ΔG5. If thedifference in binding energy is modest (not more than a few kJ/mole),then monomer association is still advantageous. This scenario ispossible and likely when known monovalent ligands or drugs areredeveloped as monomer pairs. Alternatively, when monomer pairs aredeveloped by dynamic combinatorial strategies, the selection process islikely to uncover candidates for which ΔG3>ΔG1+ΔG5 and ΔG4>ΔG2+ΔG5. Thatis, each component of the covalently linked monomer pairs will actuallybind more strongly to the target protein than individual unlinkedmonomers.

To calculate the dissociation constants K_(d3), K_(d4), and K_(d6), westart with an input set of defined K_(d1), K_(d2), and K_(d5). The total(or steady-state) concentrations of monomers C1 and C2, as well as thetotal concentration of protein target within the cell, are alsoprovided. The MatLab program (The Mathworks, Natick, Mass.) is used tosolve the following equations in two parts:

Part 1

-   Input set 1: K_(d1), K_(d2), K_(d5)-   Equation set 1: Calculation of ΔG1, ΔG2, ΔG5 in units of kJ/mole    ΔG1=−RT ln K _(d1)=−(8.3145 J/K)(10⁻³ kJ/K) (310 K)ln K _(d1)    ΔG2=−RT ln K _(d2)=−(8.3145 J/K)(10⁻³ kJ/K) (310 K)ln K_(d2)    ΔG5=−RT ln K _(d5)=−(8.3145 J/K)(10⁻³ kJ/K) (310 K)ln K _(ds)-   Input set 2: ΔG1, ΔG2, ΔG5-   Equation set 2: Calculation of ΔG3, ΔG4, ΔG6 in units of kJ/mole    ΔG3=ΔG2+ΔG5    ΔG4=ΔG1+ΔG5    ΔG6=ΔG1+ΔG2

To account for loss of entropy due to restriction of free rotation, theabove equations are corrected to account for 0 to 5 rotatable bondsbetween the monomer ligand and the linker element.ΔG3=ΔG2+ΔG5+nΔGrΔG4=ΔG1+ΔG5+nΔGrΔG6=ΔG1+ΔG2+nΔGr

Where n=total number of single bond rotations gained upon dissociationof complex C1C2T, and ΔGr=change in free energy per single bondrotation. A reasonable estimate for the value of ΔGr for rotation abouta C—C single bond is −2 kJ/mol.

-   Input set 3: ΔG3, ΔG4, ΔG6-   Equation set 3: Calculation of K_(d1), K_(d2), K_(d3)    ln K _(d3)=(−ΔG3)/(8.3145 J/K)(310 K)    ln K _(d4)=(−ΔG4)/(8.3145 J/K)(310 K)    ln K _(d6)=(−ΔG6)/(8.3145 J/K)(310 K)    or    K _(d3) =e ^(−(ΔG3)/(8.3145 J/K)(310 K))    K _(d4) =e ^(−(ΔG4)/(8.3145 J/K)(310 K))    K _(d6) =e ^(−(ΔG6)/(8.3145 J/K)(310 K))    Part 2-   Input set 4: K_(d1), K_(d2), K_(d3), K_(d4), K_(d5), K_(d6) and    starting concentration [SC₁] and [SC₂] of each monomer and the    initial concentration of the target protein [ST].

Note that the starting concentrations of each monomer and the target arenot the same as the final concentrations after equilibrium is obtained,but are related by the following equations:

$\begin{matrix}{{\lbrack T\rbrack + \left\lbrack {C_{1}T} \right\rbrack + \left\lbrack {C_{2}T} \right\rbrack + \left\lbrack {C_{1}C_{2}T} \right\rbrack} = \lbrack{ST}\rbrack} & {{Equation}\mspace{14mu} 1} \\{{\left\lbrack C_{1} \right\rbrack + \left\lbrack {C_{1}T} \right\rbrack + \left\lbrack {C_{1}C_{2}T} \right\rbrack + \left\lbrack {C_{1}C_{2}} \right\rbrack} = \left\lbrack {SC}_{1} \right\rbrack} & {{Equation}\mspace{14mu} 2} \\{{\left\lbrack C_{2} \right\rbrack + \left\lbrack {C_{2}T} \right\rbrack + \left\lbrack {C_{1}C_{2}T} \right\rbrack + \left\lbrack {C_{1}C_{2}} \right\rbrack} = \left\lbrack {SC}_{2} \right\rbrack} & {{Equation}\mspace{14mu} 3} \\{K_{d\; 1} = \frac{\left\lbrack C_{1} \right\rbrack\lbrack T\rbrack}{\left\lbrack {C_{1}T} \right\rbrack}} & {{Equation}\mspace{14mu} 4} \\{K_{d\; 2} = \frac{\left\lbrack C_{2} \right\rbrack\lbrack T\rbrack}{\left\lbrack {C_{2}T} \right\rbrack}} & {{Equation}\mspace{14mu} 5} \\{K_{d\; 3} = \frac{\left\lbrack {C_{1}T} \right\rbrack\left\lbrack C_{2} \right\rbrack}{\left\lbrack {C_{1}C_{2}T} \right\rbrack}} & {{Equation}\mspace{14mu} 6} \\{K_{d\; 4} = \frac{\left\lbrack {C_{2}T} \right\rbrack\left\lbrack C_{1} \right\rbrack}{\left\lbrack {C_{1}C_{2}T} \right\rbrack}} & {{Equation}\mspace{14mu} 7} \\{K_{d5} = \frac{\left\lbrack C_{1} \right\rbrack\left\lbrack C_{2} \right\rbrack}{\left\lbrack {C_{1}C_{2}} \right\rbrack}} & {{Equation}\mspace{14mu} 8} \\{K_{d\; 6} = \frac{\left\lbrack {C_{1}C_{2}} \right\rbrack\lbrack T\rbrack}{\left\lbrack {C_{1}C_{2}T} \right\rbrack}} & {{Equation}\mspace{14mu} 9}\end{matrix}$

These equations are linear equations and can be solved using thefollowing code in Matlab:

function [ ] = solvenonlineqn5(outfile) %Define threshold: thresh =eps;%Change this to the desired threshold; for example 1e−10; %Thiswould look for solutions with +ve real parts and%imaginary/real<threshold %Current threshold is set to2.220446049250313e−016 (2{circumflex over ( )}{−52}) load data; % Thisfile contains the data from the XLS gp = fopen(outfile,‘w’);fprintf(gp,‘T,C1,C2,C1T,C2T,C1C2,C1C2T\n’); for ctr= 1:size(data(:,1));flag = 0; syms C1T C1T C2 C2T C1C2 C1C2T; %% The equations f(1) =C1*T/C1T − K_(d1)(ctr); f(2) = C2*T/C2T − K_(d2)(ctr); f(3) =C1T*C2/C1C2T − K_(d3)(ctr); f(4) = C2T*C1/C1C2T − K_(d4)(ctr); f(5) =C1*C2/C1C2 − K_(d5)(ctr); f(6) = C1C2*T/C1C2T − K_(d6)(ctr); f(7) =C1+C1T+C1C2T+C1C2 − SC1(ctr); f(8) = C2+C2T+C1C2T+C1C2 − SC2(ctr); f(9)= T+C1T+C2T+C1C2T − ST(ctr);  list = [1 2 4 5 7 8 9]; %% Solution partS1 =solve(f(list(1)),f(list(2)),f(list(3)),f(list(4)),f(list(5)),f(list(6)),f(list(7)));outdata =[subs(S1.T),subs(S1.C1),subs(S1.C2),subs(S1.C1T),subs(S1.C2T),subs(S1.C1C2),subs(S1.C1C2T)]; for ctr2 = 1:size(outdata,1) if(length(find(real(outdata(ctr2,:))>0))==length(outdata(ctr2,:))) if(isempty(find(imag(outdata(ctr2,:))))) disp([‘Found real positivesolution for set: ’ num2str(ctr)]); parsedoutdata = outdata(ctr2,:);flag = 1; else index = find(imag(outdata(ctr2,:)));if(isempty(find(abs(imag(outdata(ctr2,index)))./abs(real(outdata(ctr2,index)))>thresh))) disp([‘Found solution with +ve real part and negligible imaginarypart for set: ’ num2str(ctr)]); parsedoutdata = real(outdata(ctr2,:));flag = 1; end end end end if flag==0 parsedoutdata = parsedoutdata*0;end %% Generating output fprintf(gp,‘%s\n’,num2str(parsedoutdata)); end fclose(gp); Note: Input set in excel file provides: K_(d1), K_(d2),K_(d3), K_(d4), K_(d5), K_(d6) and starting concentration [SC₁] and[SC₂] of each monomer and the initial concentration of the targetprotein [ST].

In these simulations, the entropy cost of binding both monomers to eachother when they are also bound to the target has been considered. When acoferon monomer binds to the target, the rotatable bonds between theligand and the linker element remain unrestricted. When two coferonmonomers bind to each other, the rotatable bonds also remainunrestricted. Only when the coferon dimer forms on the target is there aloss of free rotation, and this has an entropy cost. FIG. 42, is aschematic representation of a system of two monomers (C1, and C2) thatbind to orthogonal sites on a protein target (T) and to each other.Monomer 1 is illustrated as a green pentagon linker element tethered toa blue hexagon ligand, monomer 2 as an orange pentagon linker elementtethered to a blue hexagon ligand, and the target protein as a whiteshape. The tether represents one or more rotatable bonds between thelinker element and the ligand that binds to the target protein.

For these simulations, from 0 to 5 rotatable bonds between the linkerelements and ligands of each coferon monomer—for a total of up to 10rotatable bonds has been considered. While zero rotatable bondsrepresents a rigid architecture, and thus would be predicted to achievethe strongest binding, it also puts a considerable constraint on findingthe correct combination of linker element and ligand binding portion toachieve the precisely desired geometry for maximum binding to bothtarget and the other linker element in the coferon dimer. In contrast, aflexible connector between the linker elements and the ligands allowsfor systematic optimization of each binding event, albeit at an entropiccost that reflects the greater degree of freedom. A recent paper fromthe Whiteside group suggests that use of an oligo-ethylene oxide spacersmitigates the cost of using flexible connectors (V. M. Krishnamurthy,et. al, J. Am. Chem. Soc. 129:1312-1320 (2007), which is herebyincorporated by reference in its entirety). In the figures presented,K_(d) values placed next to (r2) and (r5) represent 2 and 5 rotatablebonds between the linker elements and ligands of each coferonmonomer—for a total of 4 and 10 rotatable bonds respectively. It ispredicted that use of an oligo-ethylene oxide or similar spacer wouldprovide K_(d) values approximately within the same range of valuesprovided.

The calculations are based on several idealized assumptions, which mayneed to be refined based on additional experimental evidence. It may bedifficult to tune the binding energies of the ligands or linker elementsto each other. Thus, when the K_(d1), K_(d2), and K_(d5) values are setfor these simulations, it is also considered that these constants mayvary three-fold. For example, monomer C1 with dissociation constantK_(d1) will be referred to the target of within a three-fold range of 1μM. Since the dissociation of the coferon dimer from the target reflectsthe contribution of at least two of these constants, the range expandsapproximately nine to ten-fold. For example, the dissociation constantof the coferon dimer from the target will be referred to within aten-fold range of about 47 pM or lower to within a ten-fold range ofabout 480 pM. As another example, if the linker elements bind to eachother via irreversible covalent bonds, the dissociation constant of thecoferon dimer from the target will be within a ten-fold range of about4.7 pM or lower to within a ten-fold range of about 48 pM.

For the steady-state calculations, influx of additional monomer underconditions where the concentration of the monomer was equal to theconcentration of the target was not corrected. Consequently, thosenumbers are artificially suppressed and result in a “dip” in the graphs.Further, steady-state simulations where target is in excess of monomerwere not corrected for the decrease in free target as additional monomermonomer enter the cell and bind to target and form dimers. Therefore,such numbers may be slightly higher than would be reflected in anexperimental system. Nevertheless, the numbers provide a rough guide ofconditions within a range that may vary about three-fold.

A few general rules arise from analysis of the data. In almost all thecases, the dissociation of one monomer from the C1-C2-target complex(K_(d3), K_(d4)) is easier than dissociation of the dimer (K_(d6))—sothat will be the preferred pathway. If one monomer binds poorly, even ifthe concentration of that monomer is 10-fold higher, that monomer willalways dissociate first, and thus binding of the dimer may not besufficiently strong enough to displace the native protein-proteininteraction. This may be observed in several examples where K_(d1) orK_(d2) is 100 μM or higher, or K_(d5) is 1,000 μM—under theconcentrations shown, more than 80% of the target is bound, but thedissociation constant is too high: FIGS. 58 and 59, K_(d3)=0.47 μM to4.8 μM, FIGS. 60 and 61, K_(d3)=0.47 μM to 4.8 μM, FIGS. 62 and 63,K_(d4)=0.47 μM to 4.8 μM, FIGS. 64 and 65, K_(d4)=0.47 μM to 4.8 μM,FIGS. 66 and 67, K_(d4)=47 nM to 480 nM, FIGS. 68 and 69, K_(d4)=47 nMto 480 nM, FIGS. 70 and 71, K_(d4)=0.12 μM to 1.2 μM, FIGS. 72 and 73,K_(d4)=0.12 μM to 1.2 μM, FIGS. 74 and 75, K_(d4)=47 nM to 480 nM, FIGS.76 and 77, K_(d4)=47 nM to 480 nM, FIGS. 78 and 79, K_(d4)=0.12 μM to1.2 μM, and FIGS. 80 and 81, K_(d4)=0.12 μM to 1.2 μM. The strength ofthe linker element binding to each other is critical to achieve thedesired enhancement of binding two adjacent ligands to the proteintarget. However, if the linker elements become associated usingessentially irreversible covalent linkage, then dissociation of thedimer (K_(d6)) is the only path and, consequently, is from 10-fold to1,000-fold stronger than the K_(d4) value when using reversible bindingbetween the linker elements.

Consider the simulation where the total concentrations of C1 and C2 are0.25 μM, the total protein concentration inside the cell is 0.1 μM,about 100,000 targets per cell (FIGS. 48 and 49). The dissociationconstants K_(d1) and K_(d2) between monomer C1 and target T, as well asbetween monomer C2 and target T are given at 1 μM. The linker elementdissociation constant, K_(d5) is given at 10 μM. The values next to (r2)and (r5) represent 2 and 5 rotatable bonds between the linker elementsand ligands of each monomer—for a total of 4 and 10 rotatable bondsrespectively. In this example, the dissociation constant between thelinker elements of the two monomers has been tuned so that at themonomer concentration of 0.1 μM each, approximately 1% of the freemonomers are in the dimer state. Although only monomers traverse thecell membrane, once inside, they bind to the target protein, whichaccelerates formation of the dimer. Dissociation of either monomer fromthe dimer bound to target (K_(d3), K_(d4)) is 47 pM and 480 pM for 2 and5 rotatable bonds, respectively. Under these starting conditions, thecoferon dimer dissociation from the target (K_(d6)) is 4.7 pM and 48 pMfor 2 and 5 rotatable bonds, respectively. If the linker elements forman irreversible covalent bond when bringing the two monomers together,then dissociation will be through K_(d6), i.e. binding will be even10-fold tighter than if the linker elements form reversible bonds. Sincethe dimer binds tightly to the protein target, even with reversiblelinker elements, it does not dissociate easily, and the majority of theprotein targets (99.7% and 97.3% for r2 and r5, respectively) are boundto achieve the desired therapeutic effect. When increasing the totalprotein concentration inside the cell to 1 μM, about 1,000,000 targetsper cell (FIGS. 50 and 51), and 10 μM, about 10,000,000 targets per cell(FIGS. 50-51), the target is saturated with dimer.

The results of these simulations for various values (in μM) of K_(d1),K_(d2), K_(d5), total monomer 1 concentration [SC1], total monomer 2concentration [SC2], total target protein concentration [ST], the numberof rotatable bonds between the linker element and the diversity elementfor each monomer (Rot Bond), and the highest K_(d) value are enumeratedin Table 4. In each instance, the highest K_(d) value (either K_(d3) orK_(d4)) is reported in the K_(d) highest column.

TABLE 4 Equilibrium dissociation constants derived from simulations ofcoferon monomer interactions with target protein. Figure# K_(d1) K_(d2)K_(d5) [SC1] [SC2] [ST] Rot Bond K_(d) highest 48 1 1 10 0.1 to 5.0 0.1to 5.0 0.1 2 47 pM 49 1 1 10 0.1 to 5.0 0.1 to 5.0 0.1 5 480 pM 50 1 110  1 to 50  1 to 50 1 2 47 pM 51 1 1 10  1 to 50  1 to 50 1 5 480 pM 5210 10 100 0.1 to 5   0.1 to 5  0.1 2 4.7 nM 53 10 10 100 0.1 to 5   0.1to 5  0.1 5 48 nM 54 10 10 100  1 to 50  1 to 50 1 2 4.7 nM 55 10 10 100 1 to 50  1 to 50 1 5 48 nM 56 10 10 100 10 to 50 10 to 50 10 2 4.7 nM57 10 10 100 10 to 50 10 to 50 10 5 48 nM 58 100 100 1000  1 to 50  1 to50 1 2 4.7 μM 59 100 100 1000  1 to 50  1 to 50 1 5 48 μM 60 100 1001000 10 to 50 10 to 50 10 2 4.7 μM 61 100 100 1000 10 to 50 10 to 50 105 48 μM NS 10 1 100 5 0.1 to 5.0 0.1 2 4.7 nM NS 10 1 100 5 0.1 to 5.00.1 5 48 nM NS 10 1 100 50  1 to 50 1 2 4.7 nM NS 10 1 100 50  1 to 50 15 48 nM NS 10 1 100 50 10 to 50 10 2 4.7 nM NS 10 1 100 50 10 to 50 10 548 nM 62 100 10 1000 50  1 to 50 1 2 0.47 μM 63 100 10 1000 50  2 to 501 5 4.8 μM 64 100 10 1000 50 10 to 50 10 2 0.47 μM 65 100 10 1000 50 11to 50 10 5 4.8 μM 66 25 25 100 25 25 1 2 47 nM 67 25 25 100 25 25 1 5480 nM 68 25 25 100 25 25 10 2 47 nM 69 25 25 100 25 25 10 5 480 nM 7025 25 1000 25 25 1 2 0.12 μM 71 25 25 1000 25 25 1 5 1.2 μM 72 25 251000 25 25 10 2 0.12 μM 73 25 25 1000 25 25 10 5 1.2 μM 74 10 10 1000 2525 1 2 47 nM 75 10 10 1000 25 25 1 5 480 nM 76 10 10 1000 25 25 10 2 47nM 77 10 10 1000 25 25 10 5 480 nM 78 100 100 250 25 25 1 2 0.12 μM 79100 100 250 25 25 1 5 1.2 μM 80 100 100 250 25 25 10 2 0.12 μM 81 100100 250 25 25 10 5 1.2 μM (K_(d1), K_(d2), K_(d5) are in μM. [SC1],[SC2], and [ST] represent the total concentration of monomer 1, monomer2, and target, respectively, and are in μM. Rot bonds indicates thenumber of rotatable bonds between the linker element and the diversityelement for each monomer. NS = Figure not shown)

Prophetic Example 6 Simulation of Binding Equilibria for Coferon Dimerto Target Protein: Determination of Concentrations of Coferon DimerBinding to a Target Protein for Various Values of Dissociation Constantsfor Both Monomers and for Reversible Association of Monomers

The simulations described in Prophetic Example 6 are performed asdescribed above in Example 5. These simulations, where the steady-stateor renewable concentrations of C1 and C2 are 0.25 μM, and the totalprotein concentration inside the cell is varied, can be extended toinclude 0.25 μM=250,000 targets, 1 μM=1,000,000 targets, 2.5μM=2,500,000 targets, and 10 μM=10,000,000 targets per cell (FIGS. 82,84, 86, 88, and 90, respectively). In all these cases, we see thedissociation of either monomer from the dimer bound to target (K_(d3),K_(d4)) is 47 pM and 480 pM for 2 and 5 rotatable bonds, respectively.Under these starting conditions, the coferon dimer dissociation from thetarget (K_(d6)) is 4.7 pM and 48 pM for 2 and 5 rotatable bonds,respectively. Since the coferon dimer binds tightly to the proteintarget, even with reversible linker elements it does not dissociateeasily, and the majority of the protein targets (ranging from 70% to100% across C1 and C2 concentrations ranging from 0.25 μM to 5 μM) arebound to achieve the desired therapeutic effect.

Note that under these dissociation values for each monomer to the target(K_(d1), K_(d2)=1 μM) or to each other (K_(d5)=10 μM)—considered weak bymost standards, the free dimer in solution is often less than 1%. Evenif monomer concentrations were brought to 25 μM in solution, thepercentage dimer would at most be 35% of the total, leaving sufficientmonomer to enter the cells. However, once the first monomer binds to thetarget, the second monomer can then also bind to the target as it nowhas the added advantage of two potential interactions (i) to the target,and (ii) to the neighboring monomers. Thus, the dissociation constant ofthe complex from the target jumps down to an impressive 0.5 to 0.05 nM.In other words, the improvement in binding affinity of a dimer over amonomer is from 2,000 to 20,000-fold. If the monomers becomeirreversibly linked, the improved binding of a dimer over a monomer isfrom 20,000 to 200,000-fold.

Consider increasing both monomer dissociation constants K_(d1) andK_(d2), such that K_(d1)=K_(d2)=10 μM, and the linker elementdissociation constant K_(d5)=10 μM, where the steady-state or renewableconcentrations of C1 and C2 are 0.25 μM, the total protein concentrationinside the cell is varied from 0.1 μM=100,000 targets to 10μM=10,000,000 targets per cell as above (FIGS. 92, 94, 96, 98, and 100,respectively). In all these cases, the dissociation of either monomerfrom the dimer bound to target (K_(d3), K_(d4)) is 0.47 nM and 4.8 nMfor 2 and 5 rotatable bonds, respectively. Under these startingconditions, the coferon dimer dissociation from the target (K_(d6)) isalso 0.47 nM and 4.8 nM for 2 and 5 rotatable bonds, respectively. Sincethe dimer binds tightly to the protein target, even with reversiblelinker elements it does not dissociate easily, and the majority of theprotein targets (ranging from 70% to 100% across C1 and C2concentrations ranging from 0.5 μM to 5 μM) are bound to achieve thedesired therapeutic effect.

Further, at the lowest C1 and C2 concentrations of 0.1 μM and 0.25 μM,the percentage of bound target increases as the target concentrationincreases, for example from 12.9% (under conditions of 5 rotatablebonds), with concentration of C1=0.1 μM, concentration of target at 0.1μM=100,000 targets, to 64.6% with concentration of target at 10μM=10,000,000 targets. In another example, bound target increases from44.4% (under conditions of 5 rotatable bonds), with concentration ofC1=0.25 μM, concentration of target at 0.1 μM=100,000 targets, to 75.7%with concentration of target at 10 μM=10,000,000 targets. Thus, undersome conditions, these coferons may be used to bind a significantlyhigher percentage of protein target present at high concentration in thetargeted cells—and thus have a greater influence on those cells—thanwhen binding to lower percentage of protein target present at lowconcentration in the non-targeted cells—and thus have lowerside-effects.

Consider altering the simulations so the monomer dissociation constantsare different, with monomer C1 dissociation constant K_(d1)=1 μM,monomer C2 dissociation constant K_(d2)=10 μM, and the linker elementdissociation constant K_(d5)=10 μM, where the steady-state or renewableconcentrations of C1 and C2 are 0.25 μM, the total protein concentrationinside the cell is varied from 0.25 μM=250,000 targets to 10μM=10,000,000 targets per cell (FIGS. 102, 104, 106, 108, and 110,respectively). In all these cases, the dissociation of monomer C2 fromthe dimer bound to target (K_(d3)) is 0.47 nM and 4.8 nM for 2 and 5rotatable bonds, respectively. Under these starting conditions, thecoferon dimer dissociation from the target (K_(d6)) is 47 pM and 480 pMfor 2 and 5 rotatable bonds, respectively. Since the dimer binds tightlyto the protein target, even with reversible linker elements it does notdissociate easily, and the majority of the protein targets (ranging from70% to 100% across C1 and C2 concentrations ranging from 0.25 μM to 5μM) are bound to achieve the desired therapeutic effect.

Further, at the lowest C1 and C2 concentrations of 0.1 μM and 0.25 μM,the percentage of bound target increases as the target concentrationincreases. The increase in percentage binding when target concentrationis higher is consistent in the context of an open system: there is aconstant influx of monomers from outside the cell to always maintain thelow concentration (i.e. 0.1 μM or 0.25 μM) inside the cell. For examplethe percentage of bound target increases from 39.7% (under conditions of5 rotatable bonds), with concentration of C1=0.1 μM, concentration oftarget at 0.1 μM=100,000 targets to 72.3% with concentration of targetat 10 μM=10,000,000 targets. Thus, under some conditions, coferons maybe used to bind a significantly higher percentage of protein targetpresent at high concentration in the targeted cells—and thus have agreater influence on those cells—than when binding to lower percentageof protein target present at low concentration in the non-targetedcells—and thus have lower side-effects.

The above simulations have been repeated for linker element dissociationconstant K_(d5)=100 μM, (For C1=1 μM, C2=1 uM, FIGS. 112, 114, 116, 118and 120, respectively; for C1=10 μM, C2=10 μM, FIGS. 122, 124, 126, 128,and 130, respectively; for C1=1 μM, C2=10 μM, FIGS. 132, 134, 136, 138,and 140, respectively).

The results of these simulations for various values (in μM) of K_(d1),K_(d2), K_(d5), total monomer 1 concentration [SC1], total monomer 2concentration [SC2], total target protein concentration [ST], the numberof rotatable bonds per monomer (Rot Bond), and the highest K_(d) valueare enumerated in Table 5. In each instance, the highest K_(d) value(either K_(d3) or K_(d4)) is reported in the K_(d) highest column. Sincethe highest K_(d) value is driven by the weakest binding monomer, the2nd monomer may have K_(d) values ranging from 100 nM up to the K_(d)value of the 1st monomer.

TABLE 5 Equilibrium dissociation constants derived from simulations ofsteady state interactions of monomers and dimers with target proteinwhere the interaction between linker elements is reversible and K_(d1)and K_(d2) vary between 1 and 10 μM, and K_(d5) varies between 10 and100 μM. Fig. # K_(d1) K_(d2) K_(d5) [SC1] [SC2] [ST] Rot Bond K_(d)highest 82 1 1 10 0.1 to 5.0 0.1 to 5.0 0.1 2 47 pM 83 1 1 10 0.1 to 5.00.1 to 5.0 0.1 5 480 pM 84 1 1 10 0.1 to 5.0 0.1 to 5.0 0.25 2 47 pM 851 1 10 0.1 to 5.0 0.1 to 5.0 0.25 5 480 pM 86 1 1 10 0.1 to 5.0 0.1 to5.0 1 2 47 pM 87 1 1 10 0.1 to 5.0 0.1 to 5.0 1 5 480 pM 88 1 1 10 0.1to 5.0 0.1 to 5.0 2.5 2 47 pM 89 1 1 10 0.1 to 5.0 0.1 to 5.0 2.5 5 480pM 90 1 1 10 0.1 to 5.0 0.1 to 5.0 10 2 47 pM 91 1 1 10 0.1 to 5.0 0.1to 5.0 10 5 480 pM 92 10 10 10 0.1 to 5.0 0.1 to 5.0 0.1 2 0.47 nM 93 1010 10 0.1 to 5.0 0.1 to 5.0 0.1 5 4.8 nM 94 10 10 10 0.1 to 5.0 0.1 to5.0 0.25 2 0.47 nM 95 10 10 10 0.1 to 5.0 0.1 to 5.0 0.25 5 4.8 nM 96 1010 10 0.1 to 5.0 0.1 to 5.0 1 2 0.47 nM 97 10 10 10 0.1 to 5.0 0.1 to5.0 1 5 4.8 nM 98 10 10 10 0.1 to 5.0 0.1 to 5.0 2.5 2 0.47 nM 99 10 1010 0.1 to 5.0 0.1 to 5.0 2.5 5 4.8 nM 100 10 10 10 0.1 to 5.0 0.1 to 5.010 2 0.47 nM 101 10 10 10 0.1 to 5.0 0.1 to 5.0 10 5 4.8 nM 102 1 10 100.1 to 5.0 0.1 to 5.0 0.1 2 0.47 nM 103 1 10 10 0.1 to 5.0 0.1 to 5.00.1 5 4.8 nM 104 1 10 10 0.1 to 5.0 0.1 to 5.0 0.25 2 0.47 nM 105 1 1010 0.1 to 5.0 0.1 to 5.0 0.25 5 4.8 nM 106 1 10 10 0.1 to 5.0 0.1 to 5.01 2 0.47 nM 107 1 10 10 0.1 to 5.0 0.1 to 5.0 1 5 4.8 nM 108 1 10 10 0.1to 5.0 0.1 to 5.0 2.5 2 0.47 nM 109 1 10 10 0.1 to 5.0 0.1 to 5.0 2.5 54.8 nM 110 1 10 10 0.1 to 5.0 0.1 to 5.0 10 2 0.47 nM 111 1 10 10 0.1 to5.0 0.1 to 5.0 10 5 4.8 nM 112 1 1 100 0.1 to 5.0 0.1 to 5.0 0.1 2 0.47nM 113 1 1 100 0.1 to 5.0 0.1 to 5.0 0.1 5 4.8 nM 114 1 1 100 0.1 to 5.00.1 to 5.0 0.25 2 0.47 nM 115 1 1 100 0.1 to 5.0 0.1 to 5.0 0.25 5 4.8nM 116 1 1 100 0.1 to 5.0 0.1 to 5.0 1 2 0.47 nM 117 1 1 100 0.1 to 5.00.1 to 5.0 1 5 4.8 nM 118 1 1 100 0.1 to 5.0 0.1 to 5.0 2.5 2 0.47 nM119 1 1 100 0.1 to 5.0 0.1 to 5.0 2.5 5 4.8 nM 120 1 1 100 0.1 to 5.00.1 to 5.0 10 2 0.47 nM 121 1 1 100 0.1 to 5.0 0.1 to 5.0 10 5 4.8 nM122 10 10 100 0.1 to 5.0 0.1 to 5.0 0.1 2 4.7 nM 123 10 10 100 0.1 to5.0 0.1 to 5.0 0.1 5 48 nM 124 10 10 100 0.1 to 5.0 0.1 to 5.0 0.25 24.7 nM 125 10 10 100 0.1 to 5.0 0.1 to 5.0 0.25 5 48 nM 126 10 10 1000.1 to 5.0 0.1 to 5.0 1 2 4.7 nM 127 10 10 100 0.1 to 5.0 0.1 to 5.0 1 548 nM 128 10 10 100 0.1 to 5.0 0.1 to 5.0 2.5 2 4.7 nM 129 10 10 100 0.1to 5.0 0.1 to 5.0 2.5 5 48 nM 130 10 10 100 0.1 to 5.0 0.1 to 5.0 10 24.7 nM 131 10 10 100 0.1 to 5.0 0.1 to 5.0 10 5 48 nM 132 1 10 100 0.1to 5.0 0.1 to 5.0 0.1 2 4.7 nM 133 1 10 100 0.1 to 5.0 0.1 to 5.0 0.1 548 nM 134 1 10 100 0.1 to 5.0 0.1 to 5.0 0.25 2 4.7 nM 135 1 10 100 0.1to 5.0 0.1 to 5.0 0.25 5 48 nM 136 1 10 100 0.1 to 5.0 0.1 to 5.0 1 24.7 nM 137 1 10 100 0.1 to 5.0 0.1 to 5.0 1 5 48 nM 138 1 10 100 0.1 to5.0 0.1 to 5.0 2.5 2 4.7 nM 139 1 10 100 0.1 to 5.0 0.1 to 5.0 2.5 5 48nM 140 1 10 100 0.1 to 5.0 0.1 to 5.0 10 2 4.7 nM 141 1 10 100 0.1 to5.0 0.1 to 5.0 10 5 48 nM (K_(d1), K_(d2), K_(d5) are in μM. [SC1],[SC2], and [ST] represent the total concentration of monomer 1, monomer2, and target, respectively, and are in μM. Rot bonds indicates thenumber of rotatable bonds between the linker element and the diversityelement for each monomer.)

Thus, a first coferon monomer C1 with dissociation constant K_(d1) ofthe diversity element from the target molecule of within a three-foldrange of from 100 nM to within a three-fold range of 1 μM, providing asecond coferon monomer C2 with dissociation constant K_(d2) of thediversity element from the target molecule of within a three-fold rangeof 1 μM, to within a three-fold range of 10 μM, with a dissociationconstant K_(d5) between the linker element of the first monomer and thelinker element of the second monomer of within a three-fold range of 10μM to within a three-fold range of 100 μM, with each monomer containingfrom 0 to 5 rotatable bonds or equivalent degrees of rotational freedombetween the linker element portion and target binding portions, withsteady-state concentrations of the coferon monomers C1 and C2 in theblood ranging from about 0.1 μM to about 5.0 μM or higher, and with theconcentration of the target protein in the target cells ranging fromabout 0.1 μM to 10 μM or higher. As a result, such dimers will achievebinding to the target, such that about 70% to 100% of the target proteinin the cells are bound by the dimer, which has a dissociation constantfrom the target molecule that is from within a ten-fold range of about47 pM or lower to within a ten-fold range of 48 nM, such that binding ofthese coferon dimers to the target protein is sufficient to displaceanother protein, protein domain, macromolecule, or substrate with anequal or higher dissociation constant from binding to the targetprotein, or is of sufficiently tight binding to activate, enhance, orinhibit the biological activity of the target protein or its bindingpartners. The monomers C1 and C2 achieve the desired therapeutic effect.

Prophetic Example 7 Simulation of Binding Equilibria for Coferon Dimersto Target Protein: Determination of Concentrations of Coferon DimersBinding to a Target Protein for Various Values of Dissociation Constantsfor Both Monomers and for Very Tight or Irreversible Association ofMonomers

The above simulations was repeated for linker element dissociationconstant K_(d5)=0.1 μM, as described in Examples 5 and 6.

The results of these simulations for various values (in μM) of K_(d1),K_(d2), K_(d5), total monomer 1 concentration [SC1], total monomer 2concentration [SC2], total target protein concentration [ST], the numberof rotatable bonds per monomer (Rot Bond), and the highest K_(d) valueare enumerated in Table 6. In each instance, the highest K_(d) value(K_(d6)) is reported in the K_(d) highest column. Since the highestK_(d) value is driven by the binding of the two monomers to the target,dissociation of the monomers from each other, i.e. K_(d5) values mayrange from 1 nM (or 0 nM for a completely irreversible covalent bond) upto the K_(d) value of the tightest binding monomer.

TABLE 6 Equilibrium dissociation constants derived from simulations ofsteady state interactions of coferon monomers and dimers with targetprotein where the interaction between linker elements is very tight orirreversible, and K_(d1) and K_(d2) vary between 1 and 100 μM. RotK_(d1) K_(d2) K_(d5) [SC1] [SC2] [ST] Bond K_(d) highest 1 1 0.1 0.1 to5.0 0.1 to 5.0 0.1 2 4.7 pM 1 1 0.1 0.1 to 5.0 0.1 to 5.0 0.1 5 48 pM 11 0.1 0.1 to 5.0 0.1 to 5.0 0.25 2 4.7 pM 1 1 0.1 0.1 to 5.0 0.1 to 5.00.25 5 48 pM 1 1 0.1 0.1 to 5.0 0.1 to 5.0 1 2 4.7 pM 1 1 0.1 0.1 to 5.00.1 to 5.0 1 5 48 pM 1 1 0.1 0.1 to 5.0 0.1 to 5.0 2.5 2 4.7 pM 1 1 0.10.1 to 5.0 0.1 to 5.0 2.5 5 48 pM 1 1 0.1 0.1 to 5.0 0.1 to 5.0 10 2 4.7pM 1 1 0.1 0.1 to 5.0 0.1 to 5.0 10 5 48 pM 1 10 0.1 0.1 to 5.0 0.1 to5.0 0.1 2 47 pM 1 10 0.1 0.1 to 5.0 0.1 to 5.0 0.1 5 480 pM 1 10 0.1 0.1to 5.0 0.1 to 5.0 0.25 2 47 pM 1 10 0.1 0.1 to 5.0 0.1 to 5.0 0.25 5 480pM 1 10 0.1 0.1 to 5.0 0.1 to 5.0 1 2 47 pM 1 10 0.1 0.1 to 5.0 0.1 to5.0 1 5 480 pM 1 10 0.1 0.1 to 5.0 0.1 to 5.0 2.5 2 47 pM 1 10 0.1 0.1to 5.0 0.1 to 5.0 2.5 5 480 pM 1 10 0.1 0.1 to 5.0 0.1 to 5.0 10 2 47 pM1 10 0.1 0.1 to 5.0 0.1 to 5.0 10 5 480 pM 10 10 0.1 0.1 to 5.0 0.1 to5.0 0.1 2 0.47 nM 10 10 0.1 0.1 to 5.0 0.1 to 5.0 0.1 5 4.8 nM 10 10 0.10.1 to 5.0 0.1 to 5.0 0.25 2 0.47 nM 10 10 0.1 0.1 to 5.0 0.1 to 5.00.25 5 4.8 nM 10 10 0.1 0.1 to 5.0 0.1 to 5.0 1 2 0.47 nM 10 10 0.1 0.1to 5.0 0.1 to 5.0 1 5 4.8 nM 10 10 0.1 0.1 to 5.0 0.1 to 5.0 2.5 2 0.47nM 10 10 0.1 0.1 to 5.0 0.1 to 5.0 2.5 5 4.8 nM 10 10 0.1 0.1 to 5.0 0.1to 5.0 10 2 0.47 nM 10 10 0.1 0.1 to 5.0 0.1 to 5.0 10 5 4.8 nM 10 1000.1 0.1 to 5.0 0.1 to 5.0 0.1 2 4.7 nM 10 100 0.1 0.1 to 5.0 0.1 to 5.00.1 5 48 nM 10 100 0.1 0.1 to 5.0 0.1 to 5.0 0.25 2 4.7 nM 10 100 0.10.1 to 5.0 0.1 to 5.0 0.25 5 48 nM 10 100 0.1 0.1 to 5.0 0.1 to 5.0 1 24.7 nM 10 100 0.1 0.1 to 5.0 0.1 to 5.0 1 5 48 nM 10 100 0.1 0.1 to 5.00.1 to 5.0 2.5 2 4.7 nM 10 100 0.1 0.1 to 5.0 0.1 to 5.0 2.5 5 48 nM 10100 0.1 0.1 to 5.0 0.1 to 5.0 10 2 4.7 nM 10 100 0.1 0.1 to 5.0 0.1 to5.0 10 5 48 nM (K_(d1), K_(d2), K_(d5) are in μM. [SC1], [SC2], and [ST]represent the total concentration of monomer 1, monomer 2, and target,respectively, and are in μM. Rot bonds indicates the number of rotatablebonds between the linker element and the diversity element for eachmonomer.)

Thus, when providing a first coferon monomer C1 with dissociationconstant K_(d1) of the diversity element from the target molecule ofwithin a three-fold range of from 1 μM to within a three-fold range of10 μM, providing a second coferon monomer C2 with dissociation constantK_(d2) of the diversity element from the target molecules of within athree-fold range of 1 μM to within a three-fold range of 100 μM, with adissociation constant K_(d5) between the linker element of the firstmonomer and the linker element of the second monomer of within athree-fold range of 1 nM (or 0 nM for a completely irreversible covalentbond) to within a three-fold range of 10 μM, with each monomercontaining from 0 to 5 rotatable bonds or equivalent degrees ofrotational freedom between the linker element portion and target bindingportions, with steady-state concentrations of the monomers C1 and C2 inthe blood ranging from about 0.1 μM to about 5.0 μM or higher, with theconcentration of the target protein in the target cells ranging fromabout 0.1 μM to 10 μM or higher, such dimers will achieve binding to thetarget. As a result, about 70% to 100% of the target protein in thecells are bound by the dimer, which has a dissociation constant from thetarget molecule that is from within a ten-fold range of about 4.7 pM orlower to within a ten-fold range of 48 nM, binding of these coferondimers to the target protein is sufficient to displace another protein,protein domain, macromolecule, or substrate with an equal or higherdissociation constant from binding to the target protein, or is ofsufficiently tight binding to activate, enhance, or inhibit thebiological activity of the target protein or its binding partners. Thecoferon monomers C1 and C2 achieve the desired therapeutic effect.

Prophetic Example 8 Characterization of Dimerization for Selected LinkerElement Precursors by Mass Spectrometry

The ability of precursors to linker elements to form dimers in aqueoussolutions was determined by mass spectrometry (MS) and massspectrometry/mass spectrometry (MS/MS). Linker elements that form dimers(or multimers) only as a result of the ionization during massspectrometry can be distinguished from those that truly form dimers inaqueous solution by determination of the fragmentation during massspectrometry/mass spectrometry.

Thus, piperidin-2-ylmethanol and cyclohexylboronic acid form a dimer(1-cyclohexylhexahydro-1H-[1,3,2]oxazaborolo[3,4-a]pyridine) in aqueoussolution. 1,3-dihydroxyacetone forms a dimer(2,5-bis(hydroxymethyl)-1,4-dioxane-2,5-diol) during ionization but inaqueous solution exists mostly as the monomer. On the other hand,2-hydroxycyclohexanone forms a dimer(dodecahydrodibenzo[b,e][1,4]dioxine-4-a,9a-diol) in aqueous solution asdetermined by MS/MS. 3-hydroxy-2-methyl-4H-pyran-4-one and zinc acetateform a dimer containing 2 molecules of 3-hydroxy-2-methyl-4H-pyran-4-oneco-ordinating one Zn cation in aqueous solution as determined by MS/MS.

Prophetic Example 9 Representative Dissociation Constants for ReversibleBoronate Formation Between Phenylboronic Acid and Selected 1,2-diols

FIG. 141 shows the equilibrium K_(d) values for a series of 1,2-diolswhen reacted with phenyl boronic acid. The K_(d) values range between206 mM for the simplest 1,2-diol, such as 2-hydroxy propanol to 10 μMfor a furanose sugar.

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow.

What is claimed:
 1. A therapeutic multimer comprising: a plurality ofcovalently or non-covalently linked monomers, each monomer comprising: adiversity element which binds to a target molecule with a dissociationconstant less than 300 μM; and a linker element, having a molecularweight less than 500 daltons, and capable of forming a reversiblecovalent bond or non-covalent tight interaction with a binding partnerlinker element on another monomer, with a dissociation constant lessthan 300 μM, with or without a co-factor, under physiologicalconditions, wherein said diversity element and said linker element arejoined together for each monomer directly or indirectly through aconnector, the plurality of monomers being reversibly covalently bondedor non-covalently linked together through their linker elements, and thediversity elements of the therapeutic multimer bind to proximatelocations of the target molecule, wherein the therapeutic multimer bindsto the target molecule with a dissociation constant of less than 10 μM,wherein said linker element is selected from the group consisting of

where R is an aliphatic or alicyclic group where Q is an aromatic,heterocyclic or nonheterocyclic ring where n=0-3, m=1-3 and p =1-2 wherethe lines crossed with a dashed line illustrate the one or more bondsformed joining the one or more diversity elements directly or through aconnector to the linker element, and wherein the binding partner linkerelement is a 1,2-diol, or a 1,3 diol, whereby said linker element andits binding partner linker element, when bound together, form 5 or 6membered boronate ester rings.
 2. The therapeutic multimer of claim 1,wherein said diversity element binds to the target molecule with adissociation constant of 100 nM to 100 μM.
 3. A therapeutic multimercomprising a plurality of combined therapeutic monomers, each monomercomprising: a diversity element which binds to a target molecule with adissociation constant less than 300 μM and a linker element, having amolecular weight less than 500 daltons, and capable of forming acovalent bond or non-covalent tight interaction with a binding partnerlinker element on another monomer, with a dissociation constant lessthan 300 μM, with or without a co-factor, under physiologicalconditions, wherein said diversity element and said linker element areconnected together directly or indirectly through a connector, for eachmonomer, the plurality of monomers forming the therapeutic multimer, andthe diversity elements for the plurality of monomers bind to proximatelocations of the target molecule, wherein said linker element is anaromatic, non-heterocyclic compound or an aliphatic compound, selectedfrom the group consisting of the Formula (B1), (B2), (B3), and (B4):

where R is an aliphatic or alicyclic group where Q is an aromatic,nonheterocyclic ring where n=0-3, m=1-3, and p=1-2 where the linescrossed with a dashed line illustrate the one or more bonds formedjoining the one or more diversity elements directly or through aconnector to the molecule of Formula (B1), (B2), (B3), and (B4), andwherein the binding partner linker element is a 1,2 diol, or a 1,3 diol,whereby said linker element and its binding partner linker element form5 or 6 membered boronate ester rings.
 4. The plurality of therapeuticmonomers of claim 3, wherein one or more of said diversity elementsbinds to the target molecule with a dissociation constant of 100 nM to100 μM.
 5. The plurality of therapeutic monomers of claim 3, wherein oneor more of said diversity elements is capable of forming a reversiblecovalent bond with the target molecule.